JP2013510576A - Methods and compositions for generation and regulation of iPS cells - Google Patents

Abstract

The present invention provides a method for generating induced pluripotent stem (iPS) cells that has higher induction efficiency than conventional methods. The method includes treating somatic cells with a nuclear reprogramming factor in combination with agents and / or p21 inhibitors that alter microRNA levels or activity in the cells. The present invention further provides iPS cells generated by such methods, as well as clinical and research uses of such iPS cells.
[Selection figure] None

Description

  The present invention relates generally to the field of induced pluripotent stem (iPS) cells, and more specifically, methods for generating such cells from somatic cells, and clinical practice of iPS cells generated by such methods. Use and research use.

  Induced pluripotent stem cells (iPSCs) display characteristics of embryonic stem (ES) cells, initially ectopic expression of four nuclear reprogramming factors (4F) in mouse somatic cells: Oct4, Sox2, Klf4 and cMyc Generated by. In human cells, in addition to the first four Yamanaka factors, iPSCs can also be generated using another set of four factors, such as Oct4 Nanog Lin28 and Sox2. Although many cell types from different tissues have been identified as reprogrammable, a low reprogramming efficiency of typically 0.01% to 0.2% is a key for iPSC induction and further therapeutic use It is a serious obstacle. A great deal of effort has been put into the development of new methods of iPSC induction, as well as screening for small molecules that increase reprogramming efficiency, but the mechanism by which primary fibroblasts are reprogrammed to an ES-like state is still large. The part is unknown.

  Various approaches have been used to understand the mechanism of cell reprogramming. In the small molecule based method, it was confirmed that the reprogramming process can be accelerated by treating cells with Dnmt1 inhibitor. TGFβ inhibition has also been found to allow faster and more efficient iPSC induction. This can replace Sox2 and cMyc. Furthermore, array analysis has shown that partially initialized iPSCs can be more fully initialized when treated with factors such as methyltransferase inhibitors. In an exhaustive genomic analysis of promoter binding and expression induction by four reprogramming factors, these factors have similar targets in iPSC and mES cells and probably regulate similar gene sets, Partial iPSCs have also been shown to change the targeting of reprogramming factors.

  More recently, several groups have identified that the p53-mediated tumor suppressor pathway can antagonize iPSC induction. Both p53 and its downstream effector p21 are induced during the reprogramming process and reduced expression of both proteins can promote iPSC colony formation. Although these proteins are up-regulated in most cells expressing four reprogramming factors (4F), cMyc has been reported to block p21 expression, but the differences between these four factors (4F) It is still unclear how in situ expression overcomes the cellular response to oncogene / transgene overexpression and why only a small population of cells is fully reprogrammed.

  MicroRNA (MicoRNA) is a single-stranded small RNA of 18-24 nucleotides that is associated with a protein complex called RNA-induced silencing complex (RISC). These small RNAs are usually generated from non-coding regions of gene transcripts and function to suppress gene expression by suppressing translation. In recent years, microRNAs have been implicated in many different important processes, including self-replicating gene expression of human ES cells, cell cycle control of embryonic stem (ES) cells, alternative splicing, and cardiac development. It was found. Furthermore, it has recently been reported that ES cell-specific microRNAs enhance mouse iPSC induction and can replace the function of cMyc during reprogramming. It has also been suggested that hES-specific miR-302 reduces the aging response by expressing four factors in human fibroblasts. However, since these microRNAs are not expressed until very near the end of the reprogramming process, it has not been known so far whether microRNAs play an important role in iPSC induction.

  The present invention is based on the critical discovery that microRNAs are required during iPSC induction. Interference with the microRNA biosynthetic mechanism results in a significant reduction in reprogramming efficiency. Highly induced microRNA clusters have been identified during the initial stages of reprogramming, and functional tests have shown that induction efficiency is increased by introducing such microRNAs into somatic cells. In addition, important regulators used for cell reprogramming have also been identified, but it is advantageous to target these regulators to significantly increase reprogramming efficiency as well as direct differentiation of iPS cells.

  Thus, in one embodiment, the present invention provides a method for generating iPS cells. The method includes contacting a somatic cell with a nuclear reprogramming factor and contacting the cell with a microRNA that alters RNA levels or activity in the cell, thereby generating an iPS cell. In one aspect, the microRNA or RNA is modified. In another aspect, the microRNA is present in a vector. In another aspect, the microRNA is present in a miR-17, miR-25, miR-106a, miR let-7 family member (eg, let-7a, miR 98) or a miR-302b cluster. In another aspect, the microRNA is miR-93, miR-106b, miR-21, miR-29a, or a combination thereof.

  In one aspect, the microRNA has a polynucleotide sequence comprising SEQ ID NO: 1. In another aspect, the microRNA has a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 2-11. In another aspect, the microRNA modulates the expression or activity of p21, Tgfbr2, p53, or a combination thereof. In another aspect, the microRNA modulates the Splay 1/2, p85, CDC42 or ERK1 / 2 pathway.

  In one aspect, the nuclear reprogramming factor is encoded by a gene contained in a vector. In another aspect, the nuclear reprogramming factor is a SOX family gene, a KLF family gene, a MYC family gene, SALL4, OCT4, NANOG, LIN28, or combinations thereof. In another aspect, the nuclear reprogramming factor is one or more of OCT4, SOX2, KLF4, C-MYC. In another aspect, the nuclear reprogramming factor comprises c-Myc. In another aspect, the induction efficiency is at least twice as compared to when no microRNA is used.

  In one aspect, the reprogramming factor is contacted with the somatic cell prior to, simultaneously with, or after contact with the microRNA. In another aspect, the somatic cell is a mammalian cell. In a further aspect, the somatic cell is a human cell or a mouse cell.

  In another embodiment, the present invention provides a method of generating iPS cells by contacting somatic cells with a nuclear reprogramming factor and an inhibitor of p21 expression or activity.

  In another embodiment, the invention contacts a somatic cell with an agent that alters RNA levels or activity within the cell, wherein the agent induces pluripotency in the somatic cell, However, the agent is not a nuclear reprogramming factor), thereby providing a method of generating induced pluripotent stem (iPS) cells by generating iPS cells. In various embodiments, the RNA is non-coding RNA (ncRNA) and includes microRNA.

  In one embodiment of the method described above, the agent is a polynucleotide, polypeptide or small molecule. In a further aspect, the polynucleotide is an antisense oligonucleotide, a chemically modified oligonucleotide, a locked nucleic acid (LNA) or DNA. In another aspect, the polynucleotide is RNA. In a further aspect, the RNA is selected from the group consisting of microRNA, dsRNA, siRNA, stRNA or shRNA. In another aspect, the somatic cell is a mouse embryonic fibroblast (MEF).

  In various aspects, an agent that alters RNA can inhibit p21, Tgfbr2, p53, or a combination thereof for expression or activity. In one aspect, the agent may be a polynucleotide, polypeptide or small molecule. In another aspect, the agent or inhibitor of p21, Tgfbr2 and / or p53 is an RNA molecule and includes microRNA, dsRNA, siRNA, stRNA or shRNA, or an antisense oligonucleotide. In an exemplary embodiment, the agent or inhibitor of p21, Tgfbr2 and / or p53 is a microRNA molecule and is encoded by a polynucleotide contained in a recombinant vector introduced into the cell.

  In various aspects, the microRNA may be a microRNA contained in a cluster that exhibits increased or decreased activity or expression during induction or differentiation of iPSCs. In one aspect, the induction efficiency is at least twice as compared to when no agent is used. In another aspect, the induction efficiency is at least 3 times that when no agent is used. In another aspect, the induction efficiency is at least 5 times that when no agent is used. In one aspect, the microRNA may be one or more microRNAs in a miR-17, miR-25, miR-106a or miR-302b cluster, miR-93, miR-106b, miR-21, miR. -29a, miR-let-7 family members (eg, let-7a; miR 98) or combinations thereof are included. In a related aspect, the microRNA is between various microRNAs (eg, those of SEQ ID NOs: 2-11) corresponding to the microRNA species in the miR-17, miR-25, miR-106a and miR-302b clusters. It has a polynucleotide sequence comprising SEQ ID NO: 1, which has been found to be conserved. In one aspect, the microRNA has a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 2-11.

  In various embodiments, the nuclear reprogramming factor is encoded by a gene contained in a recombinant vector introduced into the cell. In another aspect, the agent inhibits expression or activity of p21, Tgfbr2, p53, or a combination thereof. In another aspect, the agent modulates the Sry 1/2, p85, CDC42 or ERK1 / 2 pathway.

  In various aspects, the nuclear reprogramming factor is encoded by one or more of a SOX family gene, a KLF family gene, a MYC family gene, SALL4, OCT4, NANOG, LIN28, or combinations thereof. In an exemplary embodiment, the nuclear reprogramming factor is one or more of OCT4, SOX2, KLF4, C-MYC. In another aspect, the at least one nuclear reprogramming factor comprises c-Myc. In a further aspect, c-Myc promotes reprogramming to at least some extent by inhibiting at least one miRNA.

  In another embodiment, the present invention provides iPS cells or a population of such cells produced using the methods described herein. In another embodiment, the present invention provides an enriched population of induced pluripotent stem (iPS) cells produced by the methods described herein.

  Similarly, in another embodiment, the present invention provides differentiated cells obtained by inducing differentiation of iPSCs generated using the methods described herein. In one aspect, somatic cells are obtained by inducing differentiation by contacting iPSCs with RNA molecules or antisense oligonucleotides. In one aspect, the RNA molecule is selected from the group consisting of microRNA, dsRNA, siRNA, stRNA or shRNA.

  In another embodiment, the present invention provides a method of treating a subject using iPS cells generated using the methods described herein. The method includes inducing a somatic cell of interest into induced pluripotent stem (iPS) cells using the methods described herein, inducing differentiation of the iPS cells, and Introducing into a subject and thereby treating the condition.

  In another embodiment, the present invention provides a method for assessing the physiological function of an agent using iPS cells generated by the methods described herein or somatic cells derived therefrom. In one aspect, the method treats induced pluripotent stem (iPS) cells produced using the methods described herein and evaluates at least one change in cell function by the agent. including. In another aspect, the method treats differentiated cells obtained by inducing differentiation of the pluripotent stem cells described herein with an agent and assesses changes in cell function by the agent. Including doing.

  In another embodiment, the present invention provides a method of assessing compound toxicity using iPS cells produced by the methods described herein or somatic cells derived therefrom. In one aspect, the method includes treating induced pluripotent stem (iPS) cells produced using the methods described herein with the compound and assessing the toxicity of the compound. In another aspect, the method comprises treating differentiated cells obtained by inducing differentiation of the pluripotent stem cells described herein with a compound and assessing the toxicity of the compound.

  In another embodiment, the present invention provides a method of generating induced pluripotent stem (iPS) cells. The method includes contacting the somatic cell with at least one nuclear reprogramming factor; and contacting the cell with an inhibitor of expression or activity of p21, Tgfbr2, p53, or a combination thereof. In one aspect, the inhibitor inhibits p21 expression and / or activity. In another aspect, the inhibitor inhibits Tgfbr2 expression and / or activity. In another aspect, the inhibitor inhibits p53 expression and / or activity.

  In another embodiment, the present invention provides a method of generating induced pluripotent stem (iPS) cells. The method involves contacting a somatic cell with an agent that alters RNA levels or activity within the cell, where the agent induces pluripotency in the somatic cell, provided that the agent is Not nuclear reprogramming factors), thereby generating iPS cells.

  In another embodiment, the present invention provides a method of treating a subject. The method includes generating induced pluripotent stem (iPS) cells from a subject's somatic cells according to the methods described herein; inducing differentiation of the iPS cells; and introducing the cells into the subject. , Thereby treating the symptoms.

  In another embodiment, the present invention provides the use of microRNA to increase iPS cell production efficiency. In one aspect, the microRNA is selected from the group consisting of miR-17, miR-25, miR-93, miR-106a, miR-106b, miR-21, miR-29a, miR-302b cluster, or combinations thereof. Is done. In another aspect, the microRNA is present in a miR-17, miR-25, miR-106a or miR-302b cluster. In another aspect, the microRNA is miR-93, miR-106b, miR-21, miR-29a, or a combination thereof.

  In another embodiment, the invention provides a miR-17, miR-25, miR-93, miR-106a, miR-106b, miR-21, miR-29a, miR-302b cluster, miR let-7 family member or Providing a combination of miR sequences selected from the group consisting of those combinations. In another aspect, the microRNA is present in a miR-17, miR-25, miR-106a or miR-302b cluster. In another aspect, the microRNA is miR-93, miR-106b, miR-21, miR-29a, or a combination thereof.

It is a figure which shows involvement of the RNAi mechanism to mouse | mouth iPSC induction | guidance | derivation. Figures 1a, 1b and 1c show the knockdown of the mouse RNAi machinery genes Ago2, Drosha and Dicer by shRNA, respectively. Both mRNA and protein levels of the target gene were analyzed by RT-qPCR and shown in the histogram and corresponding Western blot. Primary factor embryonic fibroblasts (MEFs) are transduced with four factors along with shRNA targeting Drosha, Dicer and Ago2. Lentiviral shRNA is transduced into MEF together with 4 μg / μl of polybrene, and total RNA or protein is collected 3 days after transduction. The mRNA and protein levels of the target gene are analyzed by RT-qPCR and Western blotting, respectively. pLKO is an empty vector control against the shRNA lentiviral vector. pGIPZ is a lentiviral vector that expresses an untargeted shRNA. FIG. 1d shows that AGO2 knockdown reduces iPSC induction by OSK. Colonies are stained 21 days after transduction and quantified for AP. Error bars represent the standard deviation of duplicate wells. FIG. 1e shows the quantification of iPSC GFP + colonies for shAgo2. GFP + colonies are quantified 21 days after transduction. Error bars represent the standard deviation of duplicate wells. FIG. 1f shows that Ago2 knockdown dramatically reduces iPSC induction by 4F. The primary MEF is transduced with 4 reprogramming factors (OSKM (4F)) along with shRNA Ago2. Colonies can be stained for alkaline phosphatase 14 days after transduction. Alkaline phosphatase is a marker for mES / iPS cells. pLKO vector and pGIPZ vector served as negative controls. FIG. 6 shows the induction of microRNA clusters miR-17, 25, 106a and 302b during the initial phase of initialization. FIG. 2a shows a graph showing expression induction of 10 microRNA clusters in the initial stage after transduction of 4 factors. miR RT-qPCR is used to quantify expression changes in representative microRNAs of 10 clusters that are highly expressed in ES cells. MEF total RNA using MEF at the start and 4F on the 4th day after infection is analyzed. The dark bar in the histogram indicates the MEF on the fourth day after infection, and the white bar indicates the starting MEF. Asterisk indicates induced microRNA. FIG. 2b shows a seed region comparison of different miR clusters induced on day 4 after 4F transduction. Similar seed regions are underlined. FIG. 2c shows a graph for the induction of microRNA. Exemplary microRNAs can be derived using various combinations of four factors. MicroRNA expression is quantified 4 days after transduction. 4F, OSK, OS and single factors are used to analyze which factors were involved in miR expression changes. It is a figure which shows the induction enhancement of iPSC by miR-93 and miR-106b. FIG. 3a is a pictorial representation showing the time series of the initialization assay. MicroRNA mimics are transfected on day 0 and day 5 at a final concentration of 50 nM. GFP + colonies are quantified on day 11 for 4F induction and on days 15-20 for iPSC induction of OSK 3 factor. FIG. 3b is a graph showing that miR-93 and miR-106b mimics enhance iPSC induction with respect to 4F induction. Oct4-GFP MEF is transfected with 50 nM of the indicated microRNA. GFP + colonies are quantified 11 days after transduction. Induction magnification and error bars were calculated from three independent experiments using triplicate wells. FIG. 3c is a graph showing discrimination of the enhancement effect of miR-93 and miR-106b using the OSK system. MicroRNA mimics are transfected as in the 4F experiment. GFP + colonies are quantified on days 15-20. Error bars represent the standard deviation of 3 independent experiments in triplicate wells. FIG. 3d is a graph showing the effect of microRNA inhibition on reprogramming efficiency. Inhibitors of miR-93 and miR-106b dramatically reduce reprogramming efficiency. MicroRNA inhibitors are also transfected at a final concentration of 50 nM and maintain the same experimental timeline as miR mimic transfection. Error bars represent the standard deviation of 3 independent experiments in triplicate wells. It is a figure which shows the characteristic evaluation of the iPSC clone obtained from the miR mimic experiment which analyzes the expression by RT-PCR of a different endogenous ES marker. Total RNA is isolated from the iPS cell line 3 days after passage. Expression of ES cell specific markers (Eras, ECat I, Nanog and endogenous Oct4 etc.) is analyzed by RT-PCR. FIG. 6 shows targeting of mouse p21 and Tgfbr2 with miR-93 and miR-106b. FIG. 5a shows that miR-93 and 106b transfection reduces p21 protein levels. Oct4-GFP MEFs are transfected with 50 nM miR mimic and harvested 48 hours after transfection for Western analysis. Actin is used as a loading control. FIG. 5b shows that p21 is efficiently knocked down by siRNA. P21 siRNA transfected and control transfected MEFs are harvested at 48 hours and at RT-qPCR and Western blotting is performed to confirm p21 expression. p21 mRNA is normalized to GAPDH. FIG. 5c shows that iPSC induction is enhanced by knockdown of p21 by siRNA. MEF is infected with 4F virus and siRNA is transfected according to the same timeline as microRNA mimic transfection. GFP + colonies are quantified on day 11. Error bars represent at least two independent experiments with triplicate wells. FIG. 5d shows that miR-93 and 106b transfection reduces Tgfbr2 expression. Transfected cells are harvested at 48 hours for Western blotting. FIG. 5e shows that Tgfbr2 is knocked down by siRNA. Relative Tgfbr2 mRNA levels are normalized to that of Gapdh. FIG. 5f shows that iPSC induction is enhanced by knockdown of Tgfbr2 by siRNA. Error bars represent at least 3 independent experiments with 3 replicate wells. It is a figure which shows promotion of the initialization by microRNA. FIG. 6a shows that miR-17 and miR-106a can increase initialization efficiency, but miR-16 cannot increase initialization efficiency. miR-17 and miR-106a mimics are transfected into MEF at a final concentration of 50 nM. GFP + colonies are quantified 11 days after transduction. Error bars represent two independent experiments in triplicate wells. FIG. 6b shows that miR-17 and 106a target p21. Western blotting of p21 is performed 2 days after transfection of microRNA mimic into MEF. miR-17 and miR-106a target Tgfbr2 expression. MicroRNA mimics are transfected into MEF at a final concentration of 50 nM. FIG. 6c shows that miR-17 and 106a target Tgfbr2. Western blotting is performed 2 days after transfection. FIG. 6d shows a model for the role of microRNAs during iPSC induction. During the initial phase of initialization, several microRNAs are derived, including miR-17, 25 and 106a clusters. These microRNAs facilitate full reprogramming by targeting factors that antagonize the reprogramming process (such as p21 and other unidentified proteins). The rise and fall represent various potential steps and barriers during the reprogramming process, and the dashed lines indicate reprogramming barriers that are reduced by microRNA induction in the reprogrammed cells. FIG. 6 is a graph showing the dose response of miR-93 and miR-106b in mouse iPSC induction. Oct4-GFP MEFs are transfected with different concentrations of microRNA (5 nM, 15 nM and 50 nM). Mimic control siRNA is used as a control. GFP + colonies are quantified 11 days after transduction. Data represent triplicate wells in 12 well plates. FIG. 4 shows p21 expression induced during iPSC induction. FIG. 8a shows Western blot analysis using different p21 expression systems (from left to right: OSKM, OSK, OS, Klf4, cMyc and MEF controls). p21 expression is induced by Klf4 and cMyc. For Western blot analysis, MEFs infected with 4F, OSK, OS, Klf4 and cMyc are collected 5 days after transduction. FIG. 8b shows a graph showing confirmation of the expression of different transgenes in infected MEFs. It is a figure which shows inhibition by the p21 overexpression of the initialization using OSK3 factor. FIG. 9a is a graph of AP + colony quantification of iPSCs by OSK induction and p21 overexpression. Cells induced on day 21 are stained for alkaline phosphatase. The p21 virus is introduced at the same time as OSK. FIG. 9b is a graph of quantification of iPSC GFP + colonies by OSK induction and p21 overexpression. FIG. 5 shows direct regulation of p21 expression by miRNA. FIG. 10a is a pictorial representation showing two potential sites found in the p21 mRNA 3′UTR. Mutations are introduced at the first site (conservation site) to destroy the binding affinity of miR-93 and 106b. FIG. 10b is a graph showing quantification of pGL3-p21 luciferase reporter expression in Hela cells. Hela cells are harvested after 48 hours of transfection with pGL3-p21 and pRL-TK and microRNA. Results are normalized to pRL-TK levels in transfected cells. FIG. 5 shows direct regulation of Tgfbr2 expression by miRNA. FIG. 11a is a pictorial representation showing two potential sites found in the Tgfbr2 mRNA 3′UTR. FIG. 11b is a graph showing quantification of luciferase reporter expression in Hela cells, performed as in the p21 experiment. Results are normalized to pRL-TK levels in transfected cells. FIG. 6 shows relative Tgfbr2 mRNA levels in the presence of various displayed miRNAs. FIG. 3 shows that shRNA is actively expressed in shAgo2-infected MEF. FIG. 13a shows shAgo2 levels and FIG. 13b shows shRNA levels. FIG. 13c shows the expression of ES specific markers in Ago2-infected MEF. FIG. 4 shows relative miRNA expression on day 0, 4, 8, and 12 after transduction of OSKM factor. FIG. 6 shows the effect of miR-93 mimic on the relative level of miR-93. FIG. 16a shows that miR inhibitors can reduce target miR expression. FIG. 16b further illustrates the effect of miR inhibitors during the various stages of the initialization process. FIG. 4 shows promoter methylation levels at the endogenous Nanog locus when miR-93 or miR-106b is introduced. FIGS. 18a and 18b show that genes that significantly decrease after miR-93 transfection may show a 3-fold enrichment of genes that are poorly expressed in iPSC, while genes that increase after miR-93 transfection are such It shows no concentration. FIG. 19a shows the relative Tgfbr2 mRNA levels after introduction of miR-93 using either mRNA array analysis or RT-qPCR analysis. FIG. 19b shows the relative mRNA levels after introduction of miR-25, miR-93 or miR-106b. It is a figure which shows that iPS cell reprogramming efficiency increases by the inhibition of micro RNA, miR-21, and miR-29a concentrated with MEF. FIG. 20a shows that miR-29a, miR-21 and let7a are highly expressed in MEF. Total RNA is isolated from Oct4-EGFP MEF and mouse ES cells and separated by gel electrophoresis. The signal is detected using a radiolabeled probe specific for the indicated miRNA. U6 snRNA serves as a loading control. FIG. 20b shows that miRNA inhibition increases initialization efficiency. OSKM is transduced into Oct4-EGFP MEF. On the 14th day after transduction, GFP positive colonies are confirmed by fluorescence microscopy and counted. The number of GFP + colonies is normalized to the number of control treatments without anti-miR targeting and reported as fold change. Error bars represent the standard deviation of three independent experiments. ^* P value <0.05. FIG. 3 shows that c-Myc is the main repressor of miF-enriched miRNA during initialization. FIG. 21a shows Northern analysis of selected miRNAs on day 5 after initialization. Oct4-EGFP MEFs are transduced with the indicated single factor or various reprogramming factor combinations. 1F, 1 factor; 2F, 2 factors; 3F, 3 factors; OSKM: Oct4, Sox2, Klf4 and c-Myc. U6 is used as a loading control RNA. Embryonic stem cell (ES) total RNA serves as a negative control for MEFs and transduced cells. The specific miRNA displayed on the right side is detected using various probes. miR-291 blotting is a positive control for ES RNA. FIG. 21b shows a quantitative representation of miRNA expression in the presence of various reprogramming factors. Signal intensity is normalized to the intensity of U6 snRNA. The expression ratio is calculated as the expression ratio of each miRNA to the expression in MEF (this is arbitrarily set to 100%). Various miRNAs (panel A) are quantified and displayed on the right. FIG. 21c shows real-time RT-PCR analysis of selected miRNAs in Oct4-EGFP MEF at various time points after initialization with OSK or OSKM. RNA is isolated on the indicated day (D) after transduction for real-time RT-PCR analysis. Signals are normalized to U6 and shown as a percentage of miRNA expressed in MEF (this is arbitrarily set to 100). Error bars represent the standard deviation of two independent experiments. FIG. 22 shows that inhibition of miR-21 or miR-29a promotes iPS cell reprogramming by reducing p53 protein levels and upregulating the p85α and CDC42 pathways. FIG. 22a shows a Western analysis of p53, CDC42 and p85α expression after inhibition of various miRNAs. 1 × 10 ⁵ Oct4-EGFP MEF is transfected with the indicated miRNA inhibitor. Cells are harvested and analyzed after 5 days. FIG. 22b shows a quantitative representation of protein expression in the presence of the indicated miR inhibitor. Signal intensity is normalized to GAPDH intensity and is shown as a percentage of expression in control (NT) cells (this was arbitrarily set to 100). Error bars indicate the standard deviation of at least 3 independent experiments. ^* P value <0.05. FIG. 22c shows immunoblot analysis of expression of p53, CDC42 and p85α after inhibition of various miRNAs and OSKM transduction. 1 × 10 ⁵ Oct4-EGFP MEF is transfected with the indicated miRNA inhibitor. Cells are harvested after 5 days and analyzed by immunoblot. Signal intensity is normalized as described in (B). Error bars indicate the standard deviation of at least 3 independent experiments. ^* P value <0.05. FIG. 22d shows that depletion of miR-29a or p53 increases initialization efficiency. 4X10 ⁴ Oct4-EGFP MEF is transfected with the indicated siRNA and miRNA inhibitors and the OSKM reprogramming factor. GFP positive cells are counted 12 days after transduction. Error bars indicate the standard deviation of at least 3 independent experiments. ^* P value <0.05. FIG. 4 shows that miR-21 and miR-29a depletion increases initialization efficiency by down-regulating the ERK1 / 2 pathway. FIG. 23a shows a Western analysis of phosphorylated ERK1 / 2 and total ERK1 / 2 after inhibition of various miRNAs in MEF. 1 × 10 ⁵ Oct4-EGFP MEF is transfected with the indicated miRNA inhibitor, harvested 5 days later and immunoblotted. Signal intensity is normalized to actin and is shown as a percentage of the expression of the anti miR NT control. Error bars indicate the standard deviation of three independent experiments. ^* P value <0.05; ^** p value <0.005. FIG. 23b shows that depletion of miR-21 and miR-29a increases Spryl protein levels. The western blot analysis of a Spry1 expression ratio is shown. MEFs are transfected with various indicated miRNA inhibitors. Cells are harvested 5 days after transfection for Western blot analysis. Signal intensity is normalized to actin and shown as described in Figure 23a. Error bars represent the standard deviation of three independent experiments. ^* P value <0.05; ^** p value <0.005. FIG. 23c shows the rate of change in initialization efficiency after knockdown of ERK1 / 2 or GSK3β. 4X10 ⁴ Oct4-EGFP MEF is transfected with the indicated siRNA and OSKM. GFP positive cells are counted after 2 weeks. Transfection with siNT serves as a control for reprogramming efficiency. Error bars indicate the standard deviation of three independent experiments. ^** p value <0.005. FIG. 23d shows Western analysis of phosphorylated GSK-3β and total GSK-3β after inhibition of various miRNAs in MEF. 1 × 10 ⁵ Oct4-EGFP MEF is transfected with the indicated miRNA inhibitor, harvested 5 days later and analyzed by immunoblot. Signal intensity is normalized as described in Figure 23a. Error bars indicate the standard deviation of three independent experiments. It is a figure which shows that c-Myc accelerates | stimulates initialization by down-regulating miRNA, miR-21, and miR-29a concentrated with MEF. The p53 and ERK1 / 2 pathways act as initialization barriers, and miR-21 and miR-29a indirectly activate these pathways by down-regulating CDC42, p85α and Spry1. Also shown is crosstalk between the miR-21 / p53 pathway and the miR-29a / ERK1 / 2 pathway. c-Myc suppresses the expression of these miRNAs, which in turn interferes with the induction of ERK1 / 2 and p53. The dotted line shows the effect of p53 and ERK1 / 2 on iPS initialization. It is a figure which shows that inhibition of miR-21 promotes iPS cell reprogramming by OSK. Inhibitors of miRNA are introduced into Oct4-MEF during initialization with OSK. GFP positive colonies are counted at various time points after transduction. Error bars represent the standard deviation of two independent experiments. FIG. 5 shows that inhibition of miRNA does not alter apoptosis or proliferation rate during reprogramming. FIG. 26a shows that miRNA inhibitors are introduced into Oct4-MEF during initialization with OSKM. Cells are harvested 8-9 days after transduction. Apoptosis is assessed using PE Annexin V Apoptosis Detection Kit I (BD Pharmingen; catalog number 559663) and 7-amino-actinomycin (7-AAD). The signal is detected by FACS. Error bars represent the standard deviation of three independent experiments. FIG. 26b shows that miRNA inhibitors are introduced into Oct4-MEF during initialization with OSKM. Cells are harvested 8-9 days after transduction. One day prior to harvest, cells are treated with 5-ethynyl-2′-deoxyuridine (Edu) using Click-iT Edu Imaging Kits (Invitrogen; catalog number C10337). The signal is detected by FACS. Error bars represent the standard deviation of three independent experiments.

  The present invention is based on the discovery of important regulatory mechanisms involved in iPSC induction. An important aspect is the discovery of the association between cellular microRNAs with respect to iPSC induction. This is evident from observations that knockdown of important microRNA pathway proteins can lead to a significant decrease in reprogramming efficiency due to interference with the microRNA biosynthesis mechanism. In particular, miR-17-92, 106b-25 and 106a-363 have been revealed, which are at least three types of microRNA clusters that are highly induced during the early stages of reprogramming. Some microRNAs with very similar seed regions (such as miR-93 and miR-106b) greatly enhanced iPSC induction by targeting p21 expression and put the resulting clones into fully reprogrammed state Can be reached.

  According to the present invention, microRNA can directly work in iPSC induction, and the initialization efficiency is significantly reduced due to interference with the microRNA biosynthesis mechanism. The present invention provides three microRNA clusters, miR-17-92, miR-106b-25 and miR-106a-363, which are highly induced during the early stages of reprogramming. Functional analysis showed that Oct4-GFP + iPSC colony formation was promoted by introducing these microRNAs into MEF. In addition, according to the present invention, Tgfbr2 and p21 (both of which inhibit reprogramming) are directly targeted by these microRNAs, and blocking their activity results in a significant decrease in reprogramming efficiency. According to the present invention, it is proposed that miR-93 and miR-106b are important regulators of reprogramming activity.

  Before describing the present compositions and methods, it is to be understood that the invention is not limited to such compositions, methods and conditions, as the particular compositions, methods and experimental conditions described may vary. . Also, since the scope of the present invention is limited only by the appended claims, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It should also be understood.

  As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a method” includes one or more methods and / or steps of the type described herein that would be apparent to one of ordinary skill in the art upon reading this disclosure and the like. To do.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

  As discussed herein, the discovery that microRNAs are involved in the reprogramming process and iPSC induction efficiency allows the ability to significantly increase iPSC induction efficiency by manipulating the levels of these microRNAs in their cells Has reached. Thus, the present invention provides an iPS cell generation method with improved induction efficiency compared to known methods. The method involves contacting a somatic cell with a nuclear reprogramming factor and an agent that alters microRNA levels or activity within the cell (but the agent is not a nuclear reprogramming factor), whereby iPS Generating cells.

  The present invention is also based on the discovery of regulatory proteins that are directly involved in the reprogramming process and iPSC induction efficiency. One such protein is p21 (a small protein containing only 165 amino acids), which is a tumor suppressor gene during cancer development by causing p53-dependent G1 growth arrest and promoting differentiation and cellular senescence. It has been known for many years. Inhibition of p21 expression by microRNA during iPSC induction proved to increase induction efficiency in this respect. Thus, in one embodiment, the present invention provides a method of generating iPS cells by contacting somatic cells with a nuclear reprogramming factor and an inhibitor of p21 expression or activity.

  Given the involvement of RNA regulation in iPSC production, it is believed that induction can occur using agents that regulate RNA levels other than nuclear reprogramming factors. Thus, the present invention contacts somatic cells with an agent that alters RNA levels or activity within the cell (wherein the agent induces pluripotency in the somatic cell, provided that the The substance is not a nuclear reprogramming factor), thereby providing a method of generating induced pluripotent stem (iPS) cells by generating iPS cells. In various embodiments, the RNA is non-coding RNA (ncRNA), such as microRNA.

  In various embodiments, one or more nuclear reprogramming factors can be used to induce reprogramming of differentiated cells without the use of eggs, embryos or ES cells. The efficiency of the induction process is increased by utilizing agents that alter microRNA levels or activity within the cell during the induction process. Using this method, induced pluripotent stem cells having pluripotency and proliferation ability similar to those of ES cells can be established conveniently and highly reproducibly. For example, a nuclear reprogramming factor is obtained by transducing a cell with a recombinant vector containing a gene encoding a nuclear reprogramming factor together with a recombinant vector containing a polynucleotide encoding an RNA molecule (such as microRNA). Can be introduced. Thus, the cell can express a nuclear reprogramming factor that is expressed as a product of a gene contained in the recombinant vector, as well as a microRNA that is expressed as a product of the polynucleotide contained in the recombinant vector. Thereby inducing reprogramming of differentiated cells with higher efficiency (ratio) compared to using only nuclear reprogramming factors.

  As used herein, pluripotent cells are those that can divide for a long time (longer than 1 year) in vitro and are derived from all three germ layers including endoderm, mesoderm and ectoderm. Includes cells that have the unique ability to differentiate into the resulting cells.

  Somatic cells used in the present invention may be primary cells or immortalized cells. Such cells may be primary cells (non-immortalized cells) (such as freshly isolated from an animal) or may be obtained from cell lines (immortalized cells). In an exemplary embodiment, the somatic cell is a mammalian cell such as, for example, a human cell or a mouse cell. Somatic cells can be obtained from various organs (such as, but not limited to, skin, lung, pancreas, liver, stomach, intestine, heart, genitals, bladder, kidney, urethra and other urinary organs) by well-known methods. Or, in general, it may be obtained from any organ or tissue containing viable somatic cells. Examples of mammalian somatic cells useful in the present invention include adult stem cells, Sertoli cells, endothelial cells, granulosa epithelial cells, neurons, islet cells, epidermal cells, epithelial cells, hepatocytes, hair follicle cells, keratinocytes, hematopoietic cells. , Melanocytes, chondrocytes, lymphocytes (B lymphocytes and T lymphocytes), erythrocytes, macrophages, monocytes, mononuclear cells, fibroblasts, cardiomyocytes, other known muscle cells, generally any survival Examples include somatic cells. In certain embodiments, fibroblasts are used. As used herein, the term somatic cell is also intended to include adult stem cells. Adult stem cells are cells that can give rise to any cell type of a particular tissue. Examples of adult stem cells include hematopoietic stem cells, neural stem cells and mesenchymal stem cells.

  As used herein, reprogramming is intended to refer to a process that alters or reverses the differentiation state of partially or terminally differentiated somatic cells. Somatic cell reprogramming may be a partial or complete regression of the somatic differentiation state. In an exemplary embodiment, reprogramming is complete, in which case somatic cells are reprogrammed to induced pluripotent stem cells. However, initialization may be partial (such as retreating to a poorly differentiated state). For example, retreat of terminally differentiated cells to poorly differentiated cells (such as multipotent cells).

  In various aspects of the invention, the nuclear reprogramming factor is a gene that induces pluripotency, and differentiated cells or halved cells are expressed in a more primitive phenotype than the original cell (expression of pluripotent stem cells). Used to initialize the type). Such genes are utilized with agents that alter microRNA levels or activity in cells and / or inhibit p21 expression or activity to increase induction efficiency. Such genes and agents are capable of generating pluripotent stem cells from somatic cells by expression of one or more of the genes integrated into the somatic cell genome. As used herein, a gene that induces pluripotency is a gene that is related to pluripotency, and is a poorly differentiated cell from a somatic cell (pluripotent stem cell) by integration and expression of the gene. Etc.) is intended to refer to a gene capable of being generated. The expression of pluripotent genes is typically limited to pluripotent stem cells and is essential for the functional identity of pluripotent stem cells.

  One skilled in the art will appreciate that agents that alter the level or activity of microRNA in cells or inhibit the expression or activity of p21 include a variety of different types of molecules. Agents useful in any of the methods of the present invention include all types of molecules such as polynucleotides, peptides, peptidomimetics, peptoids (such as vinylogous peptoids), chemical compounds (organic molecules or Small organic molecules, etc.). Thus, in one aspect, the agent used in the methods of the invention is a polynucleotide (such as an antisense oligonucleotide or RNA molecule). In various aspects, the agent may be a polynucleotide, such as an antisense oligonucleotide or an RNA molecule (such as microRNA, dsRNA, siRNA, stRNA and shRNA). In an exemplary embodiment, the agent is a microRNA that is introduced into a cell that results in an increase in the level and activity of microRNA and / or inhibition of p21 in the cell.

  MicroRNA (miRNA) is a single-stranded RNA molecule that regulates gene expression. miRNAs are encoded by genes and transcribed from their DNA, but miRNAs are not translated into proteins; instead, each primary transcript (pri-miRNA) is processed into a short stem-loop structure called pre-miRNA, Ultimately it is processed into functional miRNA. Mature miRNA molecules are fully or partially complementary to one or more messenger RNA (mRNA) molecules and their main function is to down-regulate gene expression. MicroRNAs can be encoded by independent genes, but can also be processed (by the enzyme Dicer) from a variety of different RNA species, including introns, 3'UTRs of mRNA, long non-coding RNAs, snoRNAs and transposons . As used herein, microRNA also encompasses “mimic” microRNAs, which are external to cells that have the same or substantially the same function as their endogenous counterparts. Is intended to mean a microRNA introduced from Thus, for those skilled in the art to understand that the agent may be RNA introduced from the outside, the agent also includes compounds that increase or decrease the expression of microRNA in the cell.

  In various aspects, the microRNA may be a microRNA contained in a cluster that exhibits increased or decreased activity or expression during induction or differentiation of iPSCs. In one aspect, the microRNA is one or more microRNAs in a miR-17, miR-25, miR-106a or miR-302b cluster (such as miR-93, miR-106b, or any combination thereof). It may be. Induction of miR-17-92, miR-106b-25 and miR-106a-363 clusters has been shown to be important for correct initialization. Since such microRNAs appear to lower the initialization barrier during the process, the level of these microRNAs in their cells can be manipulated to improve initialization efficiency. Since microRNAs have been shown to be important regulatory molecules, microRNAs can also be manipulated to direct iPSC differentiation.

  It is now confirmed that three microRNA clusters are induced during iPSC induction, and several microRNAs within these clusters have been found to have the same nucleotide seed region sequence, which are similar Targeted mRNA was shown to be targeted. It has also been found that such microRNA sharing the same seed region nucleotide sequence enhances iPSC induction while reducing p21 expression. Thus, in one aspect, the microRNA is a polynucleotide sequence comprising SEQ ID NO: 1, 5′-AAUGGC, which has been found to be conserved among various microRNAs (eg, those of SEQ ID NOs: 2-11). -3 ′. Thus, in a related aspect, the microRNA has a nucleotide sequence of any of SEQ ID NOs: 2-11.

  The terms “small interfering RNA” and “siRNA” are also used herein to refer to short interfering RNA or silencing RNA, a type of short double stranded RNA molecule that plays various biological roles. Used in writing. In particular, siRNA is involved in the RNA interference (RNAi) pathway, where siRNA interferes with the expression of specific genes. In addition to their role in the RNAi pathway, siRNAs also act in RNAi-related pathways (eg, as an antiviral mechanism or in the formation of genomic chromatin structures).

  The polynucleotides of the present invention (such as antisense oligonucleotides and RNA molecules) may be of any suitable length. For example, one skilled in the art will understand what length is suitable for using an antisense oligonucleotide or RNA molecule to regulate gene expression. Such molecules typically have a length of about 5-100 nucleotides, 5-50 nucleotides, 5-45 nucleotides, 5-40 nucleotides, 5-35 nucleotides, 5-30 nucleotides, It is 25 nucleotides long, 5-20 nucleotides long or 10-20 nucleotides long. For example, the molecule is about 5 nucleotides long, 10 nucleotides long, 15 nucleotides long, 16 nucleotides long, 17 nucleotides long, 18 nucleotides long, 19 nucleotides long, 20 nucleotides long, 21 nucleotides long, 22 nucleotides long, 23 nucleotides long 24 nucleotide length, 25 nucleotide length, 26 nucleotide length, 27 nucleotide length, 28 nucleotide length, 29 nucleotide length, 30 nucleotide length, 31 nucleotide length, 32 nucleotide length, 33 nucleotide length, 34 nucleotide length, 35 nucleotide length, 40 It may be nucleotide length, 45 nucleotide length or 50 nucleotide length. Such polynucleotides may comprise at least about 15 nucleotides to more than about 120 nucleotides (at least about 16 nucleotides, at least about 17 nucleotides, at least about 18 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, at least about 21 nucleotides). Nucleotides, at least about 22 nucleotides, at least about 23 nucleotides, at least about 24 nucleotides, at least about 25 nucleotides, at least about 26 nucleotides, at least about 27 nucleotides, at least about 28 nucleotides, at least about 29 nucleotides, at least about 30 nucleotides, at least about 35 Nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleosides At least about 55 nucleotides, at least about 60 nucleotides, at least about 70 nucleotides, at least about 75 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 95 nucleotides, at least about 100 nucleotides Nucleotides, including at least about 110 nucleotides, at least about 120 nucleotides or more than 120 nucleotides).

  The term “polynucleotide” or “nucleotide sequence” or “nucleic acid molecule” is used broadly herein to mean a sequence of two or more deoxyribonucleotides or ribonucleotides linked together by phosphodiester bonds. It is done. As such, the terms include RNA and DNA (which can be a gene or portion thereof, cDNA, synthetic polydeoxyribonucleic acid sequence, etc., and can be single-stranded or double-stranded), and DNA / Includes RNA hybrids. In addition, those terms used herein can be prepared from naturally occurring nucleic acid molecules that can be isolated from cells, as well as, for example, by chemical synthesis or by enzymatic methods (such as by polymerase chain reaction (PCR)). Includes synthetic polynucleotides. For example, it should be recognized that only different terms are used for discussion purposes to distinguish different components of the composition.

  In general, nucleotides, including polynucleotides, are naturally occurring deoxyribonucleotides (such as adenine, cytosine, guanine or thymine linked to 2′-deoxyribose) or ribonucleotides (adenine, cytosine, guanine linked to ribose). Or uracil). However, depending on the use, the polynucleotide may also comprise nucleotide analogs comprising non-naturally occurring synthetic nucleotides or modified naturally occurring nucleotides. Nucleotide analogs are well known in the art and are commercially available, as are polynucleotides containing such nucleotide analogs. The covalent bond linking the nucleotides of the polynucleotide is generally a phosphodiester bond. However, depending on the purpose for which the polynucleotide is used, the covalent bond may be any of a number of other bonds, such as a thiodiester bond, a phosphorothioate bond, a peptide-like bond, or a nucleotide to produce a synthetic polynucleotide. Any other bond known to those of skill in the art useful for linking is included.

  Polynucleotides or oligonucleotides containing naturally occurring nucleotides and phosphodiester bonds can be chemically synthesized, or can be produced using recombinant DNA methods, using an appropriate polynucleotide as a template. In contrast, polynucleotides containing covalent bonds other than nucleotide analogs or phosphodiester bonds are generally chemically synthesized, but certain types of nucleotide analogs can be incorporated into polynucleotides by enzymes such as T7 polymerase. Thus, such an enzyme can be used to produce such a polynucleotide recombinantly from an appropriate template.

  In various embodiments, the antisense oligonucleotide or RNA molecule includes an oligonucleotide comprising a modification. Various modifications are known in the art and are contemplated for use in the present invention. For example, oligonucleotides containing modified backbones or unnatural internucleoside linkages are contemplated. As used herein, oligonucleotides having a modified backbone include those having a phosphorus atom in the backbone and those having no phosphorus atom in the backbone. For the purposes of this specification, as may be mentioned in the art, modified oligonucleotides that do not have a phosphorus atom in the internucleoside backbone can also be considered to be oligonucleosides.

  In various embodiments, modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl phosphonates and other alkyl phosphonates (3′-alkylene phosphonates, 5 ′ '-Alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkyl phosphoramidates), thionophosphoramidates, thionoalkylphosphonates, thios Noalkylphosphotriesters, selenophosphates and boranophosphates with conventional 3'-5 'linkages, their 2'-5' linked analogs, and one or more internucleotide linkages in 3'-3 'linkages. Include those having inverted polarity is 5'-5 'linkage or a 2'-2' linkage. Certain oligonucleotides with opposite polarity may be single 3'-3 'linkages, i.e. abasic (instead of lacking or instead of nucleobases) at the 3' most internucleotide linkages. Contains a single inverted nucleoside residue (with a hydroxyl group). Various salts, mixed salts and free acid forms are also included.

In various embodiments, a modified oligonucleotide backbone that does not contain a phosphorus atom therein is a short alkyl or cycloalkyl internucleoside bond, a mixed heteroatom and alkyl or cycloalkyl internucleoside bond, or one or more short heteroatom nucleosides. It has a main chain formed by an intermolecular bond or a heterocyclic internucleoside bond. These include morpholino linkages (partially formed from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and methylenethioformacetyl backbones Riboacetyl backbone; alkene-containing backbone; sulfamate backbone; methyleneimino and methylenehydrazino backbone; sulfonate and sulfonamide backbone; amide backbone; and others with mixed N, O, S and CH ₂ components Are included.

In various embodiments, both oligonucleotide mimetics, sugars and internucleoside linkages, ie, the backbone of nucleotide units, are replaced with novel groups. Base units are maintained for hybridization with the appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to exhibit excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of the oligonucleotide is replaced with an amide-containing backbone, in particular an aminoethylglycine backbone. The nucleobase is retained and bound directly or indirectly to the aza nitrogen atom of the main chain amide moiety. In various embodiments, the oligonucleotide can comprise an oligonucleoside comprising a phosphorothioate backbone and a heteroatom backbone. Modified oligonucleotides can also include one or more substituted sugar moieties. In some embodiments, the oligonucleotide comprises one of the following at the 2 'position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; -, S- or N- alkynyl; or O- alkyl -O--alkyl (wherein the alkyl, alkenyl and alkynyl, whether or unsubstituted are substituted _C 1 _{-C 10} alkyl or _C 2 _{-C 10} alkenyl And may be alkynyl). Particularly preferred are O [(CH ₂ ) _n O] _m CH ₃ , O (CH ₂ ) _n OCH ₃ , O (CH ₂ ) _n NH ₂ , O (CH ₂ ) _n CH ₃ , O (CH ₂ ). _n ONH ₂ and O (CH ₂ ) _n ON [(CH ₂ ) _n CH ₃ ]] _2, where n and m are 1 to about 10. Other preferred oligonucleotides include one of the following at the 2 ′ position: C ₁ -C ₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH ₃ , OCN, Cl, Br, CN, CF ₃ , OCF ₃ , SOCH ₃ , SO ₂ CH ₃ , ONO ₂ , NO ₂ , N 3, NH ₂ , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkyl Amino, substituted silyls, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacodynamic properties of oligonucleotides, and others with similar properties Substituents. Another modification includes 2'-methoxyethoxy _{(2'OCH 2 CH 2 OCH 3,} 2'-O- (2- methoxyethyl) or also known as 2'-MOE) and the like.

In a related aspect, the present invention includes the use of locked nucleic acids (LNA) to generate antisense nucleic acids with enhanced affinity and specificity for the target polynucleotide. LNA is a nucleic acid in which a 2′-hydroxyl group is attached to the 3 ′ or 4 ′ carbon atom of a sugar ring, thereby forming a bicyclic sugar moiety. The bond is preferably a methylene (—CH ₂ —) _n group (where n is 1 or 2) bridging the 2 ′ oxygen atom and the 4 ′ carbon atom.

Other modifications include 2'-methoxy _(2'-OCH 3), 2'- aminopropoxy _{(2'-OCH 2 CH 2 CH} 2 NH 2), 2'- allyl (2'-CH-CH -CH _2), 2'-O- allyl _{(2'-O-CH 2 -CH} -CH 2), 2'- fluoro (2'-F), 2'-amino, 2'-thio, 2' O-methyl, 2′-methoxymethyl, 2′-propyl and the like can be mentioned. The 2'-modification may be in the arabino (up) position or ribo (down) position. A preferred 2'-arabino modification is 2'-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly on the 3 ′ terminal nucleotide or in the 2′-5 ′ linked oligonucleotide at the 3 ′ position of the sugar and the 5 ′ position of the 5 ′ terminal nucleotide. Good. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties instead of pentofuranosyl sugars.

  Oligonucleotides can also include nucleobase modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C). And uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, adenine and 6-methyl derivatives of guanine and other Alkyl derivatives, 2-propyl derivatives of adenine and guanine and other alkyl derivatives, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and 5-halocytosine, 5-propynyluracil and 5-propynylcytosine, and pyrimidine bases Other alkynyl derivatives, 6-azouracil, 6-azocytosine and 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy And other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F -Adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido [5,4-b] [1,4] benzoxazin-2 (3H) -one), phenothiazine cytidine (1H-pyrimido [5,4-b] [1,4] benzothiazin-2 (3H) -one), substituted phenoxazine cytidine (eg, 9- (2-aminoethoxy) -H-pyrimido [5,4-b] [1 , 4] benzoxazin-2 (3H) -one), carbazole cytidine (2H-pyrimido [4,5-b] indol-2-one), pyridoindole cytidine (H-pyrimido [3 ′, 2 ′: 4,5] pyrrolo [2,3-d] pyrimidin-2-one) and the like. Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Additional nucleobases are known in the art. Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds described herein. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines (including 2-aminopropyladenine), 5-propynyluracil and 5-propynylcytosine. The 5-methylcytosine substitution is shown to increase nucleic acid duplex stability by 0.6-1.2C, and is the currently preferred base substitution, more particularly in combination with 2'-O-methoxyethyl sugar modification This is the case.

  Another modification of the antisense oligonucleotides described herein involves chemical conjugation with one or more portions or conjugates of the oligonucleotide that increase the activity, cell distribution or cellular uptake of the oligonucleotide. An antisense oligonucleotide can comprise a group of conjugates covalently linked to a functional group such as a primary or secondary hydroxyl group. The conjugate groups include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Exemplary conjugate groups include cholesterol, lipid, phospholipid, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin and dye. Groups that enhance pharmacodynamic properties include, in the context of the present invention, groups that improve oligomer uptake, enhance oligomer degradation resistance, and / or enhance sequence-specific hybridization with RNA. . Groups that enhance pharmacokinetic properties include in the context of the present invention groups that improve oligomer uptake, distribution, metabolism or excretion. Conjugate moieties include, but are not limited to, cholesterol moieties, cholic acid, thioethers such as hexyl-5-tritylthiol, thiocholesterol, aliphatic chains such as dodecanediol or undecyl residues, phospholipids, For example, dihexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamine or polyethylene glycol chain, or adamantaneacetic acid, palmityl moiety, or octadecylamine or hexylaminocarbonyl Examples include lipid moieties such as oxycholesterol moieties.

  Several genes have been found to be associated with pluripotency and suitable for use in the present invention as reprogramming factors. Such genes are known in the art, and examples thereof include SOX family genes (SOX1, SOX2, SOX3, SOX15, SOX18), KLF family genes (KLF1, KLF2, KLF4, KLF5), MYC family genes ( C-MYC, L-MYC, N-MYC), SALL4, OCT4, NANOG, LIN28, STELLA, NOBOX or STAT family genes. STAT family members can include, for example, STAT1, STAT2, STAT3, STAT4, STAT5 (STAT5A and STAT5B), and STAT6. In some cases, it is possible to use only one gene to induce pluripotency, but generally more than one gene expression is required to induce pluripotency. is there. For example, two, three, four or more genes can be simultaneously integrated into the somatic cell genome as a polycistronic construct to allow co-expression of such genes. In an exemplary embodiment, four genes are utilized to induce pluripotency, including OCT4, SOX2, KLF4 and C-MYC. Additional genes known as reprogramming factors suitable for use in the present invention are disclosed in US patent application Ser. No. 10 / 997,146 and US patent application Ser. No. 12 / 289,873, which patent applications It is made a part of this specification by quoting.

  Since all of these genes are generally present in mammals, including humans, homologs from any mammal may be used in the present invention (including but not limited to mouse, rat, cow, sheep, horse and Genes obtained from mammals including monkeys). Furthermore, in addition to the wild-type gene product, several (eg 1-10, 1-6, 1-4, 1-3 and 1 or 2) amino acid substitutions, insertions and / or Alternatively, a mutant gene product containing a deletion and having a function similar to that of the wild-type gene product can also be used. Furthermore, the combination of factors is not limited to the use of wild type genes or gene products. For example, a Myc chimera or other Myc variant can be used instead of wild-type Myc.

  The present invention is not limited to any particular nuclear reprogramming factor combination. As discussed herein, a nuclear reprogramming factor may include one or more gene products. Nuclear reprogramming factors may also include combinations of gene products, as discussed herein. Each nuclear reprogramming factor may be used alone or in combination with other nuclear reprogramming factors as disclosed herein. Furthermore, the nuclear reprogramming factors of the present invention can be identified by screening methods, for example, as discussed in US patent application Ser. No. 10 / 997,146, which is incorporated herein by reference. Part of the description. In addition, the nuclear reprogramming factor of the present invention comprises one or more factors associated with differentiation, development, proliferation, etc. and other physiologically active factors, as well as other gene products that can function as nuclear reprogramming factors. May be included.

  Nuclear reprogramming factors may include proteins or peptides. The protein may be produced from a gene, as discussed herein, or may be in the form of a fusion gene product of the protein and another protein, peptide, etc. The protein or peptide may be a fluorescent protein and / or a fusion protein. For example, a fusion protein with a green fluorescent protein (GFP) or a fusion gene product with a peptide such as a histidine tag can be used. Furthermore, by preparing and using a fusion protein with a TAT peptide obtained from viral HIV, the cellular uptake of nuclear reprogramming factor through the cell membrane can be promoted, whereby the fusion protein is added to the medium and the gene Since complicated operations such as transduction are not performed, only initialization can be induced. Since methods for preparing such fusion gene products are well known to those skilled in the art, those skilled in the art can easily design and prepare suitable fusion gene products according to the purpose.

  As discussed herein, iPSCs are induced by contacting somatic cells in combination with a nuclear reprogramming factor and an agent and / or p21 inhibitor that alters microRNA levels or activity in the cell. obtain. As will be appreciated by those skilled in the art, delivery to somatic cells may be performed by any suitable method known in the art. In one aspect, a nuclear reprogramming factor can be introduced into a cell using a recombinant vector comprising a gene encoding the nuclear reprogramming factor. Similarly, an agent that alters microRNA can be introduced into a cell using a recombinant vector comprising a polynucleotide encoding an RNA molecule (eg, microRNA, shRNA, antisense oligonucleotide, etc.). Similarly, inhibitors of p21 can be introduced into cells using recombinant vectors comprising polynucleotides encoding peptide inhibitors or RNA molecules (eg, microRNA, shRNA, antisense oligonucleotides, etc.). Thus, the cell can express a nuclear reprogramming factor that is expressed as a product of a gene contained in the recombinant vector, and similarly, an agent or p21 inhibitor as a product of the polynucleotide contained in the recombinant vector. The agent is expressed, thereby inducing the reprogramming of the differentiated cells with a higher efficiency (ratio) compared to using only the nuclear reprogramming factor.

  The nucleic acid constructs (such as recombinant vectors) of the invention can be introduced into cells using a variety of well-known techniques (such as non-viral transfection of cells). In an exemplary embodiment, the construct is incorporated into a vector and introduced into a cell to allow expression of the construct. The introduction into the cell can be any viral or non-viral transfection known in the art, including but not limited to electroporation, calcium phosphate method, nucleofection, sonoporation, heat shock, magneto Transfection, liposome method, microinjection, fine particle gun method (nanoparticle), cationic polymer method (DEAE-dextran, polyethyleneimine, polyethylene glycol (PEG), etc.) or cell fusion). Other transfection methods include proprietary transfection reagents such as Lipofectamine (TM), Dojindo Hilymax (TM), Fugene (TM), jetPEI (TM), Effectene (TM), and DreamFect (TM).

  In other embodiments, contacting a somatic cell during induction with a combination of a nuclear reprogramming factor and an agent and / or a p21 inhibitor that alters microRNA levels or activity in the cell is known in the art. Can be performed by any method known in the art, for example, direct delivery of proteins, RNA molecules, etc. through cell membranes.

  The use of nuclear reprogramming factors in combination with agents that alter microRNA levels or activity in cells and / or p21 inhibitors increases the induction efficiency compared to using reprogramming factors alone. In various aspects, induction efficiency is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% compared to conventional methods. , 300%, 400% or even 500%. For example, induction efficiency may be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% or 50% (eg, The ratio of induced cells to the total number of somatic cells at the beginning) can be as high.

  During the induction process, somatic cells may be contacted with a nuclear reprogramming factor at the same time or prior to contacting somatic cells with agents and / or p21 inhibitors that alter microRNA levels or activity in the cells. In various embodiments, the somatic cell is contacted with any other agent or inhibitor about 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 8 days, 9 days, 10 days, 11 days. The somatic cells are contacted with the reprogramming factor 12 days, 13 days, 14 days or more. In exemplary embodiments, the somatic cells are contacted with the reprogramming factor about 1, 2, 3, 4 or 5 days before contacting the somatic cell with any other agent or inhibitor.

  Further analysis may be performed to assess the pluripotency of the reprogrammed cells. Cells can be analyzed for different growth characteristics and embryonic stem cell-like morphology. For example, cells can be differentiated in vitro by adding certain growth factors known to promote differentiation into specific cell types. Reprogrammed cells that can form only a few cell types of the body are pluripotent, whereas reprogrammed cells that can form any cell type of the body are pluripotent.

  Expression profiling of reprogrammed somatic cells to assess pluripotency may also be performed. The expression of individual genes associated with pluripotency can also be investigated. In addition, the expression of embryonic stem cell surface markers can be analyzed. Detection and analysis of various genes known in the art to be associated with pluripotent stem cells include, but are not limited to, OCT4, NANOG, SALL4, SSEA-1, SSEA-3, SSEA- 4, analysis of genes such as TRA-1-60, TRA-1-81, or combinations thereof. iPS cells can express a number of pluripotent cell markers, including: alkaline phosphatase (AP); ABCG2; stage specific embryonic antigen-1 (SSEA-1); SSEA-3; SSEA-4; TRA-1-60; TRA-1-81; Tra-2-49 / 6E; ERas / ECAT5, E-cadherin (E-cadherin); β-tubulin [Alpha] -smooth muscle actin ([alpha] -SMA); fibroblast growth factor 4 (FGF4), Cripto, Daxl; zinc finger protein 296 (zinc finger protein 296) (Zfp296); N-acetyltransferase-1 (Nat1); ES cell associated transcript 1 1) (ECAT1); ESG1 / DPPA5 / ECAT2; ECAT3; ECAT6; ECAT7; ECAT8; ECAT9; ECAT10; ECAT15-1; ECAT15-2; Fthll7; Sall4; undifferentiated embryonic cell transcription factor ( Rex1; p53; G3PDH; telomerase (including TERT); silent X chromosome genes; Dnmt3a; Dnmt3b; TRIM28; F-box containing protein 15 (Fbx15); Nanog / ECAT4; Oct3 / 4; Sox2; Klf4; c-Myc; Esrrb; TDGF1; GABRB3; Zfp42, FoxD3; GDF3; CYP25A1; developmental pluripotency-associated 2 (DPPA2); T-cell lymphoma breakpoint 1 Tcl1); DPPA3 / Stella; DPPA4; as well as other common pluripotency markers, for example, any gene used during induction to initialize the cells. iPS cells can also be characterized by down-regulation of markers characteristic of differentiated cells from which iPS cells are derived.

  The invention further provides iPS cells produced using the methods described herein, as well as populations of such cells. The reprogrammed cells of the present invention that are capable of differentiating into various cell types have a variety of uses and therapeutic uses. The basic properties of stem cells, the ability to self-replicate indefinitely and the ability to differentiate into any cell type in the body, are ideal for therapeutic use.

  Thus, in one aspect, the invention further provides a method of treating or preventing a disorder and / or symptom in a subject using induced pluripotent stem cells generated using the methods described herein. The method includes obtaining somatic cells from a subject and reprogramming the somatic cells to induced pluripotent stem (iPS) cells using the methods described herein. The cells are then cultured under conditions suitable to differentiate the cells into the desired cell type suitable for treatment of the condition. The differentiated cells can then be introduced into a subject to treat or prevent symptoms.

  In one aspect, iPS cells produced using the methods described herein, as well as populations of such cells, are treated with agents that alter microRNA levels or activity in those cells. It can be differentiated in vitro by treatment or contact with such agents. Since microRNAs have been identified as important regulators in iPSC induction, it is likely that manipulation of individual microRNAs or microRNA populations can be used to direct such iPSC differentiation. Such treatment can be used in combination with growth factors or other agents and stimuli generally known in the art to promote differentiation into specific cell types.

  One advantage of the present invention is that it provides an essentially unlimited supply for isogenic or synegenic human cells suitable for transplantation. iPS cells are specifically tailored to the patient and immune rejection is avoided. Thus, significant problems associated with current transplantation methods (such as transplant rejection, which can occur due to host versus graft or graft versus host rejection) are successfully avoided. Some types of somatic cell-derived iPS cells obtained from healthy humans or fully differentiated cells prepared from iPS cells can be stored as a library of cells in an iPS cell bank, and stem cell therapy One or more iPS cells in the library can be used to prepare somatic cells, tissues or organs that do not cause rejection by the recipient.

  The iPS cells of the present invention can be differentiated into a number of different cell types for treating various disorders by methods known in the art. For example, iPS cells can be induced to differentiate into hematopoetic stem cells, muscle cells, cardiomyocytes, liver cells, chondrocytes, epithelial cells, urinary tract cells, nerve cells, and the like. The differentiated cells can then be transplanted back into the patient's body to prevent or treat symptoms. Thus, the methods of the present invention provide for myocardial infarction, congestive heart failure, stroke, ischemia, peripheral vascular disease, alcoholic liver disease, cirrhosis, where a specific cell type / tissue increase or replacement or cell dedifferentiation is desirable. Parkinson's disease, Alzheimer's disease, diabetes, cancer, arthritis, wound healing, immunodeficiency, aplastic anemia, anemia, Huntington's disease, amyotrophic lateral sclerosis (ALS), lysosomal storage disease, multiple sclerosis, spinal cord injury It can be used to treat subjects with genetic disorders and similar diseases.

  In various embodiments, the method increases the number of cells in a tissue or organ by at least about 5%, 10%, 25%, 50%, 75% or more compared to a corresponding untreated control tissue or organ. To do. In yet another embodiment, the method causes the biological activity of the tissue or organ to be at least about 5%, 10%, 25%, 50%, 75% or more relative to a corresponding untreated control tissue or organ. To increase. In yet another embodiment, the method causes angiogenesis in the tissue or organ to be at least about 5%, 10%, 25%, 50%, 75% or more relative to a corresponding untreated control tissue or organ. To increase. In yet another embodiment, the cells are administered directly to the subject at the site where an increase in cell number is desired.

  The present invention further provides methods for assessing the physiological function or toxicity of agents, compounds, pharmaceuticals, toxicants, etc. by using various cells obtained by the methods described herein.

  Somatic cells can be initialized to an ES-like state by ectopic expression of four transcription factors, Oct4, Sox2, Klf4 and cMyc, to generate induced pluripotent stem cells (iPSCs). According to the present invention, cellular microRNAs will regulate iPSC production. Knockdown of important microRNA pathway proteins can lead to a significant decrease in reprogramming efficiency. Three microRNA clusters, miR-17-92, 106b-25, and 106a-363 have been shown to be highly induced during the early stages of reprogramming. Several microRNAs with very similar seed regions (including miR-93 and miR-106b) greatly enhanced iPSC induction and inhibition of these microRNAs significantly reduced reprogramming efficiency. Furthermore, miR-iPSC clones can reach a fully initialized state. According to the present invention, Tgfbr2 and p21 will be directly targeted by these microRNAs, and siPS knockdown of both genes will actually enhance iPSC induction. Also according to the present invention, miR-93 and its family members will directly target TGF-β receptor II to promote iPSC reprogramming. According to the present invention, microRNA will act in the reprogramming process, and iPSC induction efficiency can be greatly enhanced by modulating the microRNA level in the cell.

  Induced pluripotent stem cells (iPSC) are very promising for tailor-made regenerative medicine, but the molecular basis of reprogramming is largely unknown. Overcoming barriers that maintain cell identity is an important step in reprogramming differentiated cells. Since microRNA (miRNA) modulates target genes in a tissue-specific manner, according to the present invention, different mouse embryonic fibroblast (MEF) enriched miRNAs modulate proteins that act as reprogramming barriers after transcription. It will be. Inhibition of these miRNAs should affect cell signaling and lower their barriers. According to the present invention, the initialization efficiency in MEF is increased by depletion of miR-21 and miR-29a. According to the present invention, the p53 and ERK1 / 2 pathways are regulated by miR-21 and miR-29a and will act in reprogramming. Furthermore, according to the present invention, c-Myc promotes initialization to some extent by suppressing miRNAs concentrated with MEF (such as miR-21 and miR-29a). The present invention provides miRNA function in the regulation of multiple signaling networks involved in iPSC reprogramming.

C-Myc is one of four reprogramming factors (4F: Oct3 / 4, Sox2, Klf4 and c-Myc) and plays an important role in cell proliferation and tumorigenesis. C-Myc is an important regulator of cellular emboli and apoptosis through suppression of the cyclin dependent kinase (CDK) inhibitor p21 ^Cip1 . By disabling Miz-1 function and suppressing p15 ^INK4b , c-Myc plays an important role in the immortalization of primary cells. Many transcriptional functions of c-Myc require cooperation with Max or Miz-1. Although c-Myc as a proto-oncogene greatly enhances initialization efficiency, it is not important for initialization. Therefore, defining the molecular pathway downstream of c-Myc during reprogramming can increase the therapeutic use of iPS cells without compromising reprogramming efficiency.

  Oct4-GFP mouse embryonic fibroblasts (MEF) are obtained from mice (Jackson lab, stock number 008214) with an IRES-EGFP fusion cassette at D13.5, downstream of the stop codon of pou5f1. These MEFs are cultured in DMEM (Invitrogen, 119905-065) containing 10% FBS (Invitrogen), glutamine and NEAA. In iPSC induction, only MEFs from passage 0 to passage 4 are used.

  C-Myc has been reported to have aspects that serve to maintain ES cell regeneration by regulating microRNA (miRNA) expression. MicroRNAs are 22-nucleotide non-coding small RNAs that are loaded into an RNA-induced silencing complex (RISC) and perform overall gene silencing functions. The expression of miR-141, miR-200 and miR-429 is induced by c-Myc in ES cells and antagonizes differentiation. C-Myc can also upregulate miR-17-92 microRNA clusters or by known tumor suppressor genes (such as the let-7 family, miR-15a / 16-1, miR-29 family and miR-34a) Tumor formation is also promoted by suppressing.

  Overcoming barriers mediated by factors such as Ink4-Arf, p53 and p21 that fix somatic identity is the rate-limiting step in reprogramming. Because miRNAs modulate target genes in a tissue-specific manner, according to the present invention, different MEF miRNAs will modulate proteins that act as reprogramming regulators after transcription. Inhibition of these miRNAs can affect cell signaling and lower their barriers.

  According to the present invention, depletion of abundant miRNAs (miR-21 and miR-29a) in MEF increases the initialization efficiency by about 2.1 to 2.8 times. Moreover, according to the present invention, c-Myc suppresses miRNA (miR-21 and miR-29a) and promotes initialization of MEF. Furthermore, according to the present invention, miR-21 and miR-29a regulate the p53 and ERK1 / 2 pathways by indirectly down-regulating p53 levels and ERK1 / 2 phosphorylation during the reprogramming process. It will be.

  The following examples are provided to further illustrate the embodiments of the present invention but are not intended to limit the scope of the invention. While these examples are typical of those that may be used, other procedures, methodologies, or techniques known to those skilled in the art may be used instead.

Example 1
Cell cultures, vectors and virus-transduced Oct4-GFP mouse embryonic fibroblasts (MEFs) are mice that have an IRES-EGFP fusion cassette at D13.5, downstream of the stop codon of pou5f1 (Jackson lab, stock no. 008214). ) These MEFs are cultured in DMEM (Invitrogen, 119905-065) containing 10% FBS (Invitrogen), glutamine and NEAA. In iPSC induction, only MEFs from passage 0 to passage 4 are used.

  Plasmids pMXs-Oct4, Sox2, Klf4 and cMyc are purchased from Addgene. Plasmid pMX-HA-p21 is generated by inserting N-terminal tagged p21 into the EcoRI site of the pMX vector. pLKO-shRNA clones are purchased from Open-Biosystems.

  To generate retrovirus, PLAT-E cells were seeded in 10 cm plates and the next day using 9 μg of each factor using Lipofectamine ™ (Invitrogen, 18324-012) and PLUS ™ (Invitrogen, 11514-015). Transfect. Virus is collected and combined after 2 days. For iPSC induction, MEF is seeded in a 12-well plate and the following day, 4 factor viruses are transduced with 4 μg / ml polybrene. One day after transduction, the medium is replaced with fresh MEF medium and three days later with mES culture medium supplemented with LIF (Millipore, ESG1107). On day 14 after transduction, GFP + colonies were picked and clones that were successfully expanded were cultured in DMEM containing 15% FBS (Hyclone) and LIF, thioglycerol, glutamine, and NEAA. Irradiated CF1 MEF is used as a feeder cell layer for culturing mES and the resulting iPSC clones.

  To generate the shRNA lentivirus, the shRNA lentiviral vector is co-transfected into 293FT cells with the pPACKH1 packaging system (SBI, catalog number LV500A-1). On the second day after transfection, lentivirus is collected and centrifuged at 4,000 rpm for 5 minutes at room temperature. To produce virus, 293FT cells (Invitrogen) were transfected with 4 μg pLKO or pGIPZ vector and 10 μg packaging mix in 10 cm tissue culture plates. Two days after transfection, the supernatant containing the virus was collected and used for further transduction with 4 μg / μl polybrene. ShRNA virus is added with the four factor viruses in a 1: 1: 1: 1: 1 volume ratio.

  Transfection of microRNA, siRNA and MEF is performed as follows: microRNA mimics and inhibitors, siRNA are purchased from Dharmacon. To transfect the MEF, the microRNA mimic is diluted with Opti-MEM (Invitrogen, 11058-021) to the desired final concentration. Lipofectamine ™ 2000 (Invitrogen, 11668-019) is then added to the mixture at 2 μl / well and incubated for 20 minutes at room temperature. For 12 well transfection, 80 μl of miR mix is added to each well containing 320 μl of Opti-MEM. After 3 hours, 0.8 ml of virus mix (for iPSC) or fresh medium is added to each well and the medium is replaced with fresh MEF medium the next day.

  Western blotting is performed as follows: Whole cell lysate is prepared using MPER buffer (PIERCE, 78503) on ice for 20 minutes and clarified by centrifugation at 13,000 rpm for 10 minutes. The same amount of lysate is loaded into a 10% SDS-PAGE gel and the protein is transferred to a PVDF membrane (Bio-Rad, 1620177) using a semi-dry system (Bio-Rad). The PVDF membrane is then blocked with 5% milk in TBST for at least 1 hour at room temperature or overnight at 4 ° C.

  The antibodies used include anti-p21 (BD, 556430), anti-mNanog (R & D, AF2729), anti-h / mSSEA1 (R & D, MAB2156), anti-HA (Roche, 118674233001), anti-mAgo2 (Wako, 01422023), anti-Dicer. (Abcam, ab13502), anti-Drosha (Abcam, ab12286), anti-actin (Thermo, MS1295P0), anti-AFP (Abcam, ab7751), anti-β tubulin III (R & D systems, MAB1368), anti-α-actinin (Sigma, A7811) Including.

  RT and quantitative PCR of mRNA and microRNA is performed as follows: Total RNA is extracted by the Trizol method (Invitrogen). After extraction, 1 μg of total RNA is used for RT with Superscript II ™ (Invitrogen). Quantitative PCR is performed by using Roche LightCycler 480 II ™ and Abgene's Sybrgreen mixture (Ab-4166). The primers for mouse Ago2, Dicer, Drosha, Graph and p21 are listed in Table 1 below. Other primers are described in Takahashi, K. and S. Yamanaka (2006) Cell 126 (4): 663-76.

  For quantitative analysis of microRNA, total RNA is extracted using the method described above. After extraction, 1.5-3 μg of total RNA is used for microRNA reverse transcription using the QuantMir ™ kit (SBI, RA420A-1) according to the manufacturer's protocol. The RT product is then used for quantitative PCR using the mature microRNA sequence as the forward primer and the universal primer provided in the kit.

  Immunostaining is performed as follows: Cells are washed twice with PBS and fixed with 4% paraformaldehyde for 20 minutes at room temperature. Permeabilize the fixed cells with 0.1% Triton X-100 for 5 minutes. The cells are then blocked for 1 hour at room temperature with 5% BSA in PBS containing 0.1% Triton X-100. The primary antibody is diluted 1: 100 to 1: 400 with 2.5% BSA PBS containing 0.1% Triton X-100 according to the manufacturer's recommendations. The cells are then stained with the primary antibody for 1 hour and then washed 3 times with PBS. Secondary antibody is diluted 1: 400 and cells are stained for 45 minutes at room temperature.

  Embryoid body (EB) formation and differentiation assays are performed as follows: iPS cells are trypsinized into a single cell suspension and embryonic bodies are generated using the hanging drop method. Each drop uses 4000 iPS cells in 20 μl EB differentiation medium. EBs are cultured in hanging drops for 3 days and then replated on gelatin-coated plates. After reseeding, the cells are further cultured until day 14 when regions of repeated contraction are clearly identified.

(Table 1) Primers for qPCR analysis

  Promoter methylation analysis is performed as follows: CpG methylation of Nanog and Pou5f1 promoters is analyzed according to the same procedure described previously (Takahashi, K. and S. Yamanaka (2006) Cell 126 (4): 663-76). Briefly, the genomic DNA of the resulting clone is extracted using a Qiagen ™ kit. 1 μg of DNA is then used for genome modification analysis following the manufacturer's protocol (EZ DNA Methylation-Direct kit, Zymo Research, D5020). After modification, PCR of selected regions is performed and the products are cloned into pCR2.1-TOPO ™ (Invitrogen). Ten clones are sequenced for each gene.

Example 2
Teratoma formation, chimera generation and microarray analysis Teratoma formation and chimera generation are performed as follows: To generate teratomas, iPS cells are trypsinized and resuspended at a concentration of 1 × 10 ⁷ cells / ml. Athymic nude mice are first anesthetized with avertin and then approximately 150 μl of cell suspension is injected into each mouse. Mice are examined for tumors once a week for 3-4 weeks. Tumors are harvested and fixed with zinc formalin solution at room temperature for 24 hours prior to paraffin embedding and H & E staining. To test the ability of the resulting iPSC clone to contribute to the chimera, iPS cells are injected into C57BL / 6J-Tyr ^{(C-2J) / J} (albino) blastocysts. Generally, each blastocyst receives 12-18 iPS cells. ICR recipient females are used for embryo transfer. Donor iPS cells are either agouti or black.

  mRNA microarray analysis is performed as follows: miR-93 and siControl are transfected into MEF and total RNA is harvested 48 hours after transfection. The mRNA microarray is performed at a microarray facility within the Sanford-Burnham institute. Filter gene listings for both potential functional targets (magnification> 2, p <0.05) and all targets (magnification> 25%, p <0.05) with volcano maps. Create by. The gene listing is then used for ontology analysis using GeneGo software and following the company guidelines.

The dual luciferase assay is performed as follows: The 3′UTR of both p21 and Tgfbr2 is cloned into the XbaI site of the pGL3 control vector. Each well of a 12-well plate is transfected with 200 ng of the resulting vector and 50 ng of pRL-TK (Renilla luciferase) to 1 × 10 ⁵ Hela cells seeded one day prior to transfection. Cell lysates are harvested 2 days after transfection using 50 nM microRNA for each treatment. 20 μl of the lysate is then used in a dual luciferase assay according to the manufacturer's protocol (Dual-Luciferase® Reporter Assay System Promega, E1910).

  The cell proliferation assay is performed as follows: 3000 MEFs are seeded into each well of a 96 well plate and transduced with 4F virus and shRNA lentivirus (or transfected with a microRNA inhibitor). From day 1 after transduction / transfection, every two days, cells are incubated for 1 hour in a tissue culture incubator with mES medium containing Celltiter 96 Aqueous one Solution (Promega, G3580). Absorbance at 490 nm is then measured for each well using a plate reader, the collected data is used to generate a relative growth curve, and the signal at day 1 after transduction / transfection is used as a standard.

Example 3
It was confirmed that the post-transcriptional regulatory pathway is involved in reprogramming somatic cells, and that the post-transcriptional regulatory pathway is involved in the initialization of MEF into iPS cells. To investigate the role of post-transcriptional gene regulation during iPSC induction, lentiviral shRNA vectors targeting mouse Dicer, Drosha and Ago2 are used for stable knockdown in the primary Oct4-GFP MEF. The knockdown efficiency of these shRNA constructs is verified by both Western and RT-qPCR (FIGS. 1a, 1b and 1c). Approximately 70% to 80% mRNA level knockdown is always observed with each shRNA, and a significant reduction in protein levels is also observed.

  The shRNA is then used separately to transduce MEF with a virus expressing the four factors OSKM (Oct4, Sox2, Klf4 and cMyc) at a volume ratio of 1: 1: 1: 1: 1. After 14 days, colonies are fixed and stained for alkaline phosphatase (AP) activity, which is a widely used ES cell marker. AP + colonies are quantified for each treatment, and knockdown of the key RNAi machinery proteins Dicer, Drosha and Ago2 dramatically reduces AP + colonies compared to pLKO and pGIPZ controls. Similar results are observed using OSK (3 Factor 3F) transduction.

  Quantification of both GFP + and AP + colonies confirmed that Ago2 knockdown dramatically reduced reprogramming efficiency while not affecting the growth of transduced fibroblasts. (FIGS. 1d, 1e and 1f). Despite reduced initialization efficiency due to Ago2 knockdown, some GFP + colonies with shAgo2 are infected MEFs, and in further characterization these colonies are positive for shRNA integration, where shRNA is actively It was found to be expressed (FIGS. 13a and 13b). These cells also expressed all of the ES-specific markers tested, but displayed at the endogenous Oct4 locus (FIG. 13c). These data strongly suggest that post-transcriptional regulation, especially microRNAs, play an important role in the reprogramming process.

Example 4
MicroRNA clusters are induced during somatic cell reprogramming It has been found that microRNA miR-17, 25, 106a and 302b clusters are induced during the early stages of reprogramming. Since the four transcription factors induce many gene expression changes during iPSC induction, these factors induce several ES-specific microRNAs and these factors help in the successful initialization of MEF I guess it can. Although the latest publications on ES-specific microRNAs that enhance iPSC induction support the hypothesis, the reported microRNAs were not found until very near the end of reprogramming. By analyzing the published results, nine microRNA clusters known to be highly expressed in mouse ES cells were selected for analysis and are shown in Table 2.

  Two representative microRNAs from each cluster were evaluated using a miR qPCR-based method to determine different reprogramming stages (Day 0, Day 4, Day 8 and Day 12 after OSKM factor transduction). Quantify expression changes in day). Many ES-specific microRNAs (such as miR-290 and miR-293 clusters) are not induced until day 8 (FIG. 14), and at this stage GFP + colonies can already be detected. Several other microRNA clusters (including miR-17-92, 25-106b, 106a-363 and 302b-367) are expressed to varying degrees by day 4 after transduction of the four factors. (FIG. 2a). Among these four microRNA clusters, the level of miR-302b-367 in MEF is the lowest. Among the three clusters that are highly induced on day 4 of initialization, there is some sharing of seed regions that are very similar (FIG. 2b), and they act in initialization, It suggests that it can be targeted.

(Table 2) List of microRNAs used in iPSC experiments

  An analysis is then performed to determine which of the four factors are involved in the induction of these microRNAs. Total RNA is harvested 4 days post-infection for miR qPCR analysis by transducing MEF with various combinations of four factors at the same dose (Figure 2c). This analysis confirms that cMyc alone can induce the expression of miR-17-92, miR-25-106b and miR-106a-363 clusters. However, in all cases, the combination of all four reprogramming factors induces the most abundant expression of the microRNA cluster, and its strong expression correlates with maximum reprogramming efficiency (FIG. 2c).

  These results indicate that three microRNA clusters (including miR-17-92, 25-106b, 106a-363) are induced during the early stages of initialization, and that the expression of these microRNAs is Although it was most highly induced by the four factor sets, it was confirmed that even a single factor could induce their expression to a lesser extent.

Example 5
MicroRNAs enhance iPSC induction MicroRNAs miR-93 and miR-106b have been found to enhance mouse iPSC induction. The four identified microRNA clusters contain several microRNAs with similar seed regions, so the miR-106b-25 cluster is composed of three microRNAs (ie, miR-25, miR-93 and This cluster is further analyzed since it contains miR-106b). miR-93 and miR-106b have the same seed region, and both are highly induced by four reprogramming factors (FIG. 2a). If these microRNAs function in reprogrammed cells, introduction of these microRNAs during the process would be expected to improve the efficiency of iPSC induction.

  The functional testing of these induced microRNAs uses a strategy of directly transfecting microRNA mimics into MEF (FIG. 3a). MicroRNAs were introduced twice with 4 factor (or OSK) viruses on days 0 and 5 and a reporter MEF that expresses GFP under the control of the endogenous Oct4 promoter was used. For example, on day 0 and day 5, microRNA mimics are combined with a vector expressing either all four factors (4F, OSKM) or a single Oct4, Sox2 and Klf4 (OSK). Transfect MEFs carrying GFP directly and assay reprogramming based on GFP expression. If these cells are successfully initialized to iPSC, they become GFP positive (+). GFP + colonies are quantified around day 11 to assess reprogramming efficiency (Figure 3b; Table 3). In fact, transfection of miR-93 and miR-106b mimic resulted in an approximately 4-6 fold increase in GFP + colonies in both 4F and OSK transduction (FIG. 3c), and these induced during iPSC induction. MicroRNA was confirmed to promote MEF reprogramming.

^＊ 12ウェルプレート(ゼラチンコーティング処理)に4x10⁴個MEF/ウェル (Table 3) Number of GFP + colonies by miR for iPSC induction

^* 4x10 ⁴ MEF / well in 12 well plate (gelatin coating)

  Dose-dependent experiments show that initialization efficiency is improved with miRs as low as 5-15 nM (FIG. 7). When colonies are stained with alkaline phosphatase substrate, there appears to be no significant increase in AP + colonies with miR mimic transfection, suggesting that miR-93 and miR-106b can promote the maturation process of iPSC colonies. This is a phenomenon observed with the OSK system (in miR mimic-transfected cells, many GFP + colonies appear on day 15 after OSK transduction, whereas in control wells, iPSC colonies matured at this stage are completely absent. (Not shown).

  To confirm that these microRNAs are important in iPSC induction, miR inhibitors are also used to knock down targeted microRNAs during the process. All of the tested miR inhibitors can efficiently reduce target miR expression and their transfection does not affect proliferation (FIGS. 16a and 16b). Consistent with miR mimic experiments, knockdown of miR-93 and miR-106b can drive a dramatic decrease in GFP + colonies (FIG. 3d). Although miR-25 mimics do not enhance iPSC induction of MEF, knockdown of this microRNA reduces initialization efficiency by approximately 40% (FIG. 3d), and miR-25 may also act during the initialization process. It is suggested. As a control, the Let7a inhibitor did not have any effect on reprogramming efficiency. These data strongly indicate that miR-93 and miR-106b facilitate the initialization of MEF to iPSC. Initialization efficiency can be further increased by modulating these microRNAs during iPSC induction.

Example 6
In order to investigate whether induced clones induced by microRNA reach full pluripotency state, obtain several iPSC clones for each microRNA as well as miR control, Analyzes the expression of pluripotency markers. All clones are GFP + (showing Oct4 expression to be reactivated). Immunostaining confirmed that Nanog and SSEA1 were also activated in all clones. RT-qPCR for other mES markers (such as Eras, ECat I and endogeneous Oct4) show similar results. Whole genome mRNA expression profiling also shows that the resulting clones show a gene expression pattern more similar to mouse ES cells than MEF. Promoter methylation at the endogenous Nanog locus was analyzed and all tested clones showed a demethylated promoter as observed in mouse ES cells (FIG. 17).

  To investigate whether the resulting clones show full differentiation potential of mES cells, embryoid body (EB) formation is evaluated. All of the resulting clones show efficient EB formation, which shows positive staining for lineage markers such as β-tubulin III (ectoderm), AFP (endoderm) and α-actinin (mesoderm). EBs that repeat contraction are also obtained from these cells, indicating that functional cardiomyocytes can be derived from these miR-iPSC clones. When these miR-iPSCs are injected into athymic nude mice, teratomas are easily induced in 3-4 weeks. Finally, as a more stringent test, miR-induced iPSC clones are injected into albino / black B6 blastocysts to generate chimeric mice. Furthermore, these cells could contribute to the genital ridge of the resulting E13.5 embryo. These results indicate that the miR-93 and miR-106b promoting effect on reprogramming does not alter the differentiation potential of induced pluripotent cells and the resulting clones differentiate into all three germline types Show that you can.

Example 7
To further understand the mechanism underlying miR-93 and miR-106b- enhanced reprogramming efficiency that targets Tgfbr2 and P21 in mice, miR-93 and miR-106b investigate the cellular targets of these microRNAs . Since miR-93 shares the same seed region as miR-106b, it is first selected for analysis. miR-93 mimic is transfected into MEF and total RNA is collected on day 2 for mRNA expression profile analysis. The analysis identifies potential functional targets for miR-93 compared to published MEF and iPSC expression profiles. Genes that were significantly reduced by miR-93 transfection showed a three-fold enrichment of genes that are poorly expressed in iPSCs (FIG. 18a), whereas genes increased by miR-93 transfection show such enrichment. Not shown. In addition, pathway ontology analysis is performed on the expression profile of miR-93 transfected MEF. Interestingly, two important pathways of iPSC induction are regulated by the miR-93: TGF-β signaling pathway and the G1 / S transition pathway.

  In TGF-β signaling, one of the most markedly reduced genes by miR-93 transfection is Tgfbr2. Tgfbr2 is a constitutively active receptor kinase that plays an important role in TGF-β signaling and a recent small molecule screen enhances its heterodimeric partner, Tgfbr1, an inhibitor of iPSC It has been shown. MicroRNA target site prediction indicates that miR-93 and its family of microRNAs have two conserved targeting sites in their 3′UTR. Therefore, miR-93 is selected as the first target candidate for further investigation.

  Regarding G1 / S transition, recent results in human solid tumor samples (breast, colon, kidney, stomach and lung) and gastric cancer cell lines indicate that miR-106b-25 cluster is a cell cycle regulator (CDK inhibitors p21 and p57). P21 is selected as a potential target because it has been shown that human and mouse p21 share the conserved miR-93 / 106b target site of the 3′UTR.

  In addition, mouse ES cell-specific microRNA clusters (including miR-290 and miR-293 clusters) have also been shown to target several G1-S phase transition negative regulators (including p21). In addition, miR-290 and 293 cluster microRNAs share a seed region very similar to miR-93 and miR-106b. Therefore, p21 is also analyzed as a target candidate. Furthermore, p21 is greatly induced by four factors OSKM during the initial stage of iPSC induction (FIG. 8a). Detailed analysis has revealed that the induction of p21 is mainly due to overexpression of Klf4 and cMyc, since the combination of Oct4 and Sox2 does not show significant changes in p21 levels (FIG. 8a).

  To confirm whether mouse Tgfbr2 and p21 are targeted by miR-93 and miR-106b, miR mimics are transfected into MEF and whole cell lysates are analyzed 48 hours later by Western blotting. In fact, miR-93 and miR-106b effectively reduced both Tgfbr2 and p21 protein levels (FIGS. 5a and 5d), and further reduced p21 mRNA levels by about 25-30%, and Tgfbr2 mRNA levels. Is reduced by about 60-70% (FIG. 19). To further investigate whether p21 is a direct target of miR-93 and miR-106b, a luciferase assay (a luciferase reporter with a p21 3′UTR sequence is inserted downstream of the firefly luciferase coding sequence) is performed. The luciferase assay has shown that consistent about 40% suppression of luciferase activity can be achieved by transfecting miR mimics into Hela cells. It has also been found that microRNA mimic repression can be completely abolished when mutations are introduced into the seed region of the conserved p21 3′UTR target site (FIG. 10). For Tgfbr2, the luciferase assay also showed an approximately 50% reduction in GL activity, while the miR-93 mutant had no such effect (FIG. 11).

  Epigenetic modifications required for reprogramming can be inhibited by cell cycle arrest promoted by p21, because such modifications are more likely to occur in proliferating cells. To determine whether p21 expression interferes with iPSC induction, HA-tagged p21 cDNA is cloned into the pMX retrovirus backbone and overexpressed in MEF cells. When HA-p21 virus is introduced into MEF along with four OSKM factors, almost complete inhibition of iPSC induction is observed based on alkaline phosphatase staining and Oct4-GFP positive colony formation (FIG. 9a). Similar results are obtained when using three OSK factors for initialization (FIG. 9b).

  Since analysis has shown that miR-93 and miR-106b efficiently suppress both Tgfbr2 and p21 expression, whether both Tgfbr2 and p21 can antagonize reprogramming Investigate further. Tgfbr2 or p21 siRNA are transfected into MEFs using the same experimental time series used for microRNA mimics. Western blotting and RT-qPCR confirm that siRNA efficiently knocks down both protein and mRNA levels, respectively, without viral transduction (FIGS. 5b and 5e). Next, MEF initialization is initiated by OSKM transduction and Oct4-GFP + colonies are quantified 11 days after transduction. An induction of approximately twice the number of colonies is observed for each gene (FIGS. 5c and 5f). At the same time, our data confirm that Tgfbr2 and p21 are direct targets of miR-93 and miR-106b, and down-regulation of these genes can facilitate the reprogramming process.

Example 8
The pluripotency miR-93 and 106b of iPSC clones obtained from miR-93 and miR-106b transfection were confirmed for their ability to enhance mouse iPSC induction, but the induced cells were in a fully pluripotent state The question remains whether or not. To answer this question, several iPSC clones are obtained for each microRNA and miR control and analyzed for pluripotency markers and differentiation potential. These resulting clones are all GFP + (indicating reactivation of the Oct4 locus). It was also confirmed by immunostaining that Nanog and SSEA1 were also activated in these cells. RT-qPCR for other mES markers shows similar results. The whole genome mRNA expression profile also shows that these resulting clones have a gene expression pattern very similar to mES but not MEF. Promoter methylation of the endogenous Oct4 and Nanog loci was also analyzed and all tested clones were observed to have a demethylated promoter.

  To investigate whether the resulting clones have the full differentiation potential of mES cells, embryonic body formation is used as an initial test. All of the resulting clones yielded efficient EB formation and these EBs have been found to be positive for lineage marker staining. EBs that repeat contraction are also obtained from these cells.

  Finally, as a more rigorous test, these resulting clones are injected to see if they contribute to chimeric mice. In practice, chimeras are obtained from all tested clones. These results indicate that the miR-93 and miR-106b promoting action on reprogramming does not change the ability of the induced cells, and that induced clones that have reached an ES-like state can differentiate into all three strains. Show what you can do.

Example 9
As discussed herein that up-regulation of other microRNAs also enhances iPSC induction , three microRNA clusters were identified to be induced by four factors during iPSC induction, and within these clusters Several microRNAs have been found to have the same seed region and they have been shown to target similar mRNAs (FIG. 2). To investigate whether other microRNAs sharing the same seed region as miR-93 and miR-106b could enhance iPSC induction as well, miR-17 and miR-106a microRNA mimics were Test using a test procedure similar to that described above for miR-93 mimic treatment and iPSC induction. In fact, these microRNAs promoted reprogramming similar to that seen in the miR-106b-25 cluster (FIG. 6a), and all these miR transfections reduced Tgfbr2 and p21 protein levels. Occurs (FIGS. 6b and 6c).

  At the same time, these results indicate that the induction of miR-17-92, miR-106b-25 and miR-106a-363 clusters is important for accurate reprogramming, and up-regulation of these microRNAs is the process of iPSC generation This suggests lowering the initialization barrier to (Fig. 6d). Thus, the intracellular level of these microRNAs can be manipulated to improve initialization efficiency.

Example 10
iPSC reprogramming mechanism The resulting clones are known to activate endogenous Oct4-GFP expression. Colonies are picked from day 12 after OSKM transduction with microRNA mimic and maintained on irradiated MEF feeder plates. Green fluorescence can be observed as a GFP signal at the endogenous pou5f1 locus. Clones can be shown using ES specific markers alkaline phosphatase staining and immunostaining (based on Nanog and SSEA1 staining). Hoechst 33342 can be used for nuclear staining. Cells from all three germ layers can be obtained in an embryoid body (EB) assay using induced iPSC clones. IPS cells were cultured in approximately 4000 cells / 20 μl droplets for EB formation for 3 days, and then EB was gelatin coated for further culturing until days 12-14 where repeated contraction cardiomyocytes are observed Reseeding on plates. Cells can be immunostained with different lineage markers (including ectoderm marker β-tubulin III; endoderm marker AFP; and mesoderm marker α-actinin). Teratomas can be generated by injection of iPS cells, 1.5 million cells are injected into each mouse, and tumors are harvested 3-4 weeks after injection for paraffin embedding and H & E staining. Chimeric mice can also be produced using induced clones. iPS cells are injected into blastocysts of albino or black C57B6 mice (NCI) and iPSC contribution can be seen by agouti or black coat color.

  Day 12 reprogrammed cells can be stained with alkaline phosphatase substrate. According to the present invention, miR mimic transfection does not cause a significant increase in AP + colonies, but knockdown of miR-93 and 106b results in a significant decrease in GFP + colonies as well as AP + colonies. MicroRNA mimics do not affect overall AP + colony formation, while inhibitors do.

  Since the discovery that MEF can be reprogrammed into iPS cells, much effort has been devoted to understanding the basic mechanisms of this wonderful process. The results described herein have confirmed for the first time that post-transcriptional gene regulation is directly involved during reprogramming and that reprogramming efficiency can be significantly altered by interference with the RNAi machinery. In addition, as shown in the previous examples, the three microRNA clusters are significantly up-regulated by the four factors used to induce iPS cells, and the microRNA in these clusters is at least Two important pathways are probably targeted: TGF-β signaling and cell cycle control.

  While this study is ongoing, several recent reports have also confirmed that the p53 pathway, including several downstream tumor suppressor genes (such as p21), is one of the major barriers during iPSC induction. There is also plenty of evidence to show that ectopic expression of four factors (OSKM) readily upregulates p53 and initiates a continuous response in cell defense programs (cell cycle arrest, apoptosis or DNA damage response, etc.) is there. These defense responses are probably at the base of low initialization efficiency (considered around 0.1%). However, these data do not explain how successfully reprogrammed cells can overcome cell barriers and become iPS cells. The examples described herein allow these cells to overcome barriers in at least some but not all by inducing the expression of microRNAs that target pathways that antagonize successful reprogramming. Is shown. By modulating microRNA levels in primary fibroblasts, a significant increase in reprogramming efficiency can be achieved.

  TGF-β signaling is an important pathway that functions in diverse processes such as gastrulation, organ-specific morphogenesis, and tissue homeostasis. With current models of standard TGF-β transduction, the TGF-β ligand binds to TGF-β receptor II (Tgfbr2), which then forms a heterodimer with Tgfbr1 to form receptor-related Smads. Has been shown to transmit signals. TGF-β signaling has been reported to function in both human and mouse ES cell self-renewal, and FGF2 is a growth factor that is widely used in ES cell cultures and is responsible for TGF-β ligand expression. Induces and suppresses BMP-like activity. Blocking TGF-β receptor I family kinases with chemical inhibitors interferes with ES cell self-renewal. These discoveries are particularly important for iPSC induction because such inhibitors appear to play a completely different role during reprogramming. Recent chemical screens have shown that small molecule inhibitors of TGF-β receptor I (Tgfbr1) can actually enhance iPSC induction and replace Sox2 requirements by inducing Nanog expression. Furthermore, treatment of cells during reprogramming with TGF-β ligand has an adverse effect on iPSC induction. Thus, TGF-β signaling is important for ES cell self-renewal but is a barrier to reprogramming. According to the present invention, in addition to Tgfbr1, the activity of the constitutively active kinase Tgfbr2 will also antagonize reprogramming. Also according to the present invention, miR-93 and its family members will target Tgfbr2 directly to modulate its signaling and reprogramming.

  P21 (which is a small protein containing only 165 amino acids) has long been recognized as a tumor suppressor gene during cancer development by causing p53-dependent G1 growth arrest and promoting differentiation and cellular senescence. According to the present invention, p21 expression is up-regulated when four types of factors (OSKM) are introduced into MEF cells, but iPSC induction is almost completely blocked by overexpression of p21 (FIG. 9). This upregulation would antagonize the initialization process (FIG. 8). Since the Klf4 reprogramming factor binds to the p21 promoter and increases p21 transcription, the induction of p21 in cells undergoing reprogramming may be dependent on p53 but may not be dependent on p53.

  This raises an interesting question about the function of the four reprogramming factors, since the same transcription factor may promote iPSC induction and may antagonize iPSC induction. In fact, there is currently no evidence that can rule out the possibility that a certain level of p21 induction is beneficial to the initialization process. In addition to the well-known role in p53-dependent cell cycle arrest, p21 has also been reported to have some oncogenic activity. For example, p21 has oncogenic activity by protecting cells from apoptosis, a function unrelated to its normal function in cell cycle control.

  The potential benefit of p21 in reprogramming may depend on its ability to regulate gene expression through protein-protein interactions. For example, p21 can directly bind to several proteins involved in apoptosis, such as caspase 8, caspase 10, and procaspase 3. In another example, p21 is also a suppressor of Myc's pro-apoptotic effect by binding to the Myc N-terminus and blocking Myc-Max heterodimer formation. In fact, when Myc itself is overexpressed in MEF, there is a marked increase in cell death in the cell culture, whereas in cells transduced with the four factors, cell death is only in myc samples. Compared to the minimum. Thus, induction of p21 can be a barrier to the reprogramming process, but can improve reprogramming efficiency because it can maintain a certain level of p21 required to reduce cell apoptosis.

  Since transfection of miR-93 and miR-106b has a higher effect on reprogramming than p21 siRNA transfection, miR-93 and 106b did not suppress p21 expression as much as p21 siRNA. The data shown can be considered as partial evidence to support this hypothesis. However, this effect may be due to the targeting of multiple proteins including Tgfbr2 and p21 by these microRNAs.

  Since microRNAs typically target multiple cellular proteins, the stimulatory effects of miR-93 and miR-106b provide an opportunity to find additional genes involved in reprogramming and the process is better understood. In fact, in addition to p21, several other genes that have been reported to be negative regulators of the G1-S phase transition are also miR-93 and miR-106b targets in the 3′UTR region of mRNA transcripts. It has a site (Rb1, Rbl1, Rbl2, and Lats2 etc.). Another reported interesting target of miR-93 and miR-106b is the transcription factor E2F1, which is often found to be deregulated and overactivated in many human tumor samples. One critical function of E2F1 is to activate expression of the CDKN2A locus encoding ARF and INK4a. The Ink4a / Arf locus can also inhibit reprogramming efficiency. Thus, according to the present invention, miR-93 and miR-106b transfections can target E2F1, reduce the likelihood of activating the CDKN2A locus, and lower the initialization barrier.

  Finally, miR-17-92, miR-106b-25, and miR-106a-363 clusters are completely conserved between mice and humans. Therefore, according to the present invention, the promoting effect of miR-93 and miR-106b can be applied to human initialization.

Example 11
MicroRNA modulates iPS cell reprogramming Mouse embryonic fibroblast (MEF) induction: Oct4-EGFP MEF is a mouse strain B6; 129S4-Pou5f1 ^{tm2 (EGFP) Jae} / J (Jackson laboratory; stock number 008214) From the WiCell Laboratory website (www.wiCell.org/). Oct4-EGFP MEFs are maintained in 0.1% gelatin coated plates with MEF complete medium (10% FBS, nonessential amino acids, L-glutamine containing, DPM without sodium pyruvate).

Initialization with retrovirus: ⁴ × 10 ⁴ Oct4-EGFP MEFs are transduced with pMX retrovirus to misexpress Oct4, Sox2, Klf4 and c-Myc (Addgene). Two days later, transduced Oct4-EGFP MEF was supplied with ES medium (DMEM containing 15% ES-screened FBS, non-essential amino acids, L-glutamine, monothioglycerol and 1000 U / ml LIF) every other day. Change. Unless otherwise noted, approximately 2 weeks after transduction, reprogrammed stem cells (defined as EGFP + iPSC colonies) are scored by fluorescence microscopy. To obtain iPSCs, EGFP + colonies are manually collected under a stereomicroscope (Leica).

MicroRNA inhibitors or siRNA transfections: Inhibitors of let-7a, miR-21, and miR-29a microRNAs are purchased from Dharmacon. ⁴ × 10 ⁴ Oct4-EGFP MEFs are transfected with Lipofectamine and inhibitors according to manufacturer's instructions (Invitrogen). After 3-5 hours, the medium is discarded and replaced with MEF complete medium; for initialization, retrovirus encoding reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) is added and the medium is replaced with complete medium the next day. Unless otherwise noted, inhibitors or siRNA are reintroduced 5 days after transfection / transduction.

For Northern analysis, 1 × 10 ⁵ Oct4-EGFP MEFs are transfected and harvested 5 days later. Total RNA is isolated by TRIZOL (Invitrogen) and approximately 9 μg of total RNA is separated on a 14% denaturing polyacrylamide gel (National Diagnostics). RNA is transferred onto Hybond-XL membrane (GE healthcare), and microRNA is detected with a specific DNA probe labeled with an isotope. The signal intensity is visualized with a phosphoimager and analyzed using Multi Gauge V3.0 (FUJIFILM). MicroRNA signal intensity is normalized to that of U6 snRNA. Experiments are performed in triplicate.

For Western analysis, 1 × 10 ⁵ Oct4-EGFP MEFs are transfected and harvested 5 days later. Total protein is prepared with M-PER buffer (Pierce) and the same amount of total protein is separated on a 10% SDS-PAGE gel. The protein is transferred to a PVDF membrane and bands are detected using the following antibodies: GAPDH (Santa Cruz; catalog number sc-20357), p53 (Santa Cruz; catalog number sc-55476), PI3 kinase p85 (Cell Signaling; Catalog number 4257); Cdc42 (Santa Cruz; catalog number sc-8401); p-ERK1 / 2 (Cell Signaling; catalog number 9101); ERK1 / 2 (Cell Signaling; catalog number 9102); p-GSK3β (Cell Signaling; Catalog number 9323); GSK3β (Cell Signaling; catalog number 9315); β-actin (Thermo Scientific; catalog number MS-1295). Signal intensity is quantified by Multi Gauge V3.0 (FUJIFILM) and normalized to GAPDH or β-actin. The experiment is repeated 3-5 times.

In vitro differentiation and teratoma formation assay: For in vitro differentiation, iPSCs were dissociated with trypsin / EDTA and final concentration in embryoid body (EB) medium (DMEM containing 15% FBS, non-essential amino acids, L-glutamine) Resuspend at 5 × 10 ⁴ cells / ml. To induce EB formation, 1000 iPS cells in 20 μl are cultured in a hanging drop with an inverted Petri dish lid. After 3-5 days, EBs are harvested and transferred to 6-well plates with 0.1% gelatin coating at approximately 10 EBs per well. Two weeks after EB formation, cardiomyocytes (mesoderm) that repeat contraction are confirmed by a microscope. Cells obtained from endoderm and ectoderm were confirmed by α-fetoprotein (R &D; catalog number MAB1368) antibody and neuron-specific βIII-tubulin (abcam; catalog number ab7751) antibody, respectively.

In the teratoma assay, 1.5 × 10 ⁶ iSPCs are trypsinized, resuspended in 150 μl, and then injected subcutaneously into the dorsal hind limb of an athymic nude mouse anesthetized with avertin. Three weeks later, the mice are sacrificed and teratomas are collected. Tumor masses are fixed, dissected, and analyzed in the Cell Imaging-Histology core facility at the Sanford Burnham Laboratory.

  Chimeric analysis: iPSC medium is changed 2 hours before harvesting. Trypsinized iPSCs are incubated in 0.1% gelatin coated plates for 30 minutes to remove feeder cells. iPSCs are injected into E3.5 C57BL / 6-cBrd / cBrd blastocysts and then transferred to pseudopregnant recipient females. After birth, the contribution of iPSC is evaluated by the hair color of the child: black is derived from iPSC.

  Immunofluorescence and alkaline phosphatase (AP) staining: Seed and culture in 6-well plates coated with iPSC 0.1% gelatin. After 4 days, cells are fixed with 4% paraformaldehyde (Electron Microscopy Sciences; catalog number 15710-S). In immunofluorescence staining, fixed cells are permeabilized with 0.1% Trixton X-100 in PBS and blocked with 5% BSA / PBS. Antibodies against SSEA-1 (R &D; catalog number MAB2155) and Nanog (R &D; catalog number AF2729) are used as ES markers. Nuclei are visualized by Hoechst 33342 staining (Invitrogen). For AP staining, fixed cells are treated with alkaline phosphatase substrate according to the manufacturer's instructions (Vector Laboratories; catalog number SK-5100).

Example 12
Inhibition of miR-21 or miR-29a enhances reprogramming efficiency Mouse embryonic fibroblast (MEF) induction: Oct4-EGFP MEF is a mouse strain B6; 129S4-Pou5f1 ^{tm2 (EGFP) Jae} / J (Jackson laboratory; From stock number 008214), using the protocol provided on the WiCell Laboratory website (www.wiCell.org/). Oct4-EGFP MEFs are maintained in 0.1% gelatin coated plates with MEF complete medium (10% FBS, nonessential amino acids, L-glutamine containing, DPM without sodium pyruvate).

  To determine if inhibition of MEF-specific miRNA lowers the reprogramming barrier, MEF-enriched miRNAs are analyzed and their levels compared to those seen in mouse ES (mES) cells. As shown in FIG. 20a, let-7a, miR-21 and miR-29a are highly expressed in MEF compared to mES cells. On the other hand, miR 291 is very abundant in mES but absent in MEF (FIG. 20a). Next, for let-7a, miR-21, and miR-29a, miRNA inhibitors were combined with Oct4-EGFP MEF (Oct4- It is introduced into the MEF carrying the EGFP reporter. On day 14 after transduction, cells treated with miR-21 inhibitors show an approximately 2.1-fold improvement in reprogramming efficiency compared to non-targeting (NT) controls (FIG. 20b). Similarly, after inhibition of miR-29a, reprogramming efficiency is significantly improved about 2.8-fold (FIG. 20b). A minor effect on OSKM reprogramming is observed after let-7a inhibition, following similar antagomir treatment used for miR 29a or 21 inhibition (FIG. 20b). To further test whether miRNA inhibition promotes reprogramming by three factors in the absence of c-Myc, miRNA inhibitors were administered to cells with OSK that reprograms cells with much lower efficiency than OSKM. Transduce. The number of iPS cell colonies reprogrammed with OSK increases in the presence of miR-21 inhibitor compared to treatment with OSK alone (FIG. 25). These results indicate that depletion of miRNAs miR-21 and miR-29 enriched in MEF significantly promotes 4F reprogramming, and miR-21 blockade of reprogramming by 3 factors (OSK). Shows a moderate improvement in efficiency.

  C-Myc represses miRNA let-7a, miR-16, miR-21, miR-29a and miR-143 expression during reprogramming: Recent studies have shown that OSKM factors have epigenetic mechanisms and transcription It has been shown to alter cell identity through both mechanisms. According to the present invention, the OSKM reprogramming factor will be able to down-regulate MEF enriched miRNA. To evaluate the potential impact of each reprogramming factor on miRNA expression, MEFs are transduced with various combinations of OSKM factors and subjected to Northern blot analysis (FIG. 21a). Interestingly, only Sox2 induces miR-21, miR-29a and let-7a expression levels higher than 2-fold compared to the MEF control (FIG. 21b, left panel). Klf4 has a mild but similar effect to Sox2 on the selected miRNA (FIG. 21b, left panel). Only with Oct4 overexpression, miRNAs do not change expression levels (Figure 21b, left panel). In contrast to Oct4, Sox2 and Klf4, the single factor c-Myc down-regulates the expression of miR-21 and miR-29a (the most abundant miRNA in MEF) by about 70% of the MEF control (FIG. 21a). And FIG. 21b, left panel). Furthermore, among the various combinations of the two factors (2F) shown in FIG. 21b (middle panel), the combination comprising c-Myc is all three miRNAs (miR-21, miR-29a and let- 7a) can be increased by about 25-80% (FIG. 21b, middle panel). Sox2 and Oct4 increase miR-21 and miR-29a 1.5 and 2.3 fold over MEF controls, as well as the effect of 1F on miRNA, and OK and SK have an effect on miRNA expression There is no significant impact. Furthermore, between various combinations of the three factors (3F), miRNA-21 expression is reduced by about 70% and 78% in SKM cells and OKM cells, respectively, compared to the expression seen in MEFs, Similarly, miR-29a expression is reduced by approximately 48-70% with the 3F combination containing c-Myc (FIG. 21b, right panel). Inclusion of c-Myc in the 3F combination also slightly reduces the let-7a level (Figure 21b, right panel). OSK without c-Myc had little effect on miRNA expression (Figure 21b, right panel). Thus, these results strongly suggest that c-Myc plays an important role in the regulation of miRNA expression during reprogramming.

  To further confirm that c-Myc is the first factor that antagonizes miRNA expression, OSK was transduced into cells with or without c-Myc, and miRNA expression was varied after transduction. At various time points, it is investigated by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). In contrast to OSK, expression of let-7a, miR-16, miR-21, miR-29a, miR-143 during initialization by OSKM transduction is greatly reduced (FIG. 21c), and c-Myc is MEF. It can be seen that it plays a central role in regulating the expression of concentrated miRNAs, including the most abundant ones, let-7a, miR-21 and miR-29a. These data also suggest that c-Myc promotes reprogramming to some extent through miRNA down-regulation.

Example 13
iPS cells induced by miRNA depletion acquire pluripotency According to the present invention, mouse iPS cells obtained using miR-21 and miR-29a inhibitors will be pluripotent. OSKM / anti miR-29a iPS cells can be stained with ES cell markers. For further analysis, GFP + colonies obtained after treatment with OSKM and miR-29a inhibitor are picked. Representative colonies expressing the embryonic stem cell markers Nanog and SSEA1 are identified. Endogenous Oct4 is also activated, which can be shown by EGFP staining. Strong alkaline phosphatase (AP) activity can be observed as one of the ES markers.

In vitro differentiation of OSKM / anti miR-29a iPS cells can be performed. Embryoid bodies can be formed in vitro and cultured for 2 weeks. Cells can be fixed and stained with anti-alpha fetoprotein (for mesoderm) and anti-beta tubulin III (for ectoderm). Nuclei can be observed as counterstained by Hoescht staining. OSKM / anti miR-29a iPS cell teratoma formation analysis can also be performed. 1.5 × 10 ⁶ iPSCs are injected subcutaneously into athymic nude female mice. Tumor mass is harvested 3 weeks after injection and fixed for histopathological analysis. Various tissues from three germ layers can be identified, including intestinal-like epithelium (endoderm), adipose tissue, cartilage and muscle (mesoderm), and nerve tissue and epidermis (ectoderm) It is. Chimeric analysis of OSKM / anti miR-29a iPS cells and OSK / anti miR-21 iPS cells can also be performed. 8-14 iPS cells can be injected into E3.5 mouse blastocysts. The contribution of iPS cells to each chimera can be estimated by evaluating the black hair color and can be observed as a percentage.

  In order to investigate whether blockade of miR-21 or miR-29a interferes with iPS cell pluripotency, iPS cells using OSKM / anti miR-29a or OSK / anti miR-21 were pluripotent. To evaluate. First, cells are harvested manually approximately 2 weeks after initialization and expanded to examine morphology and expression of ES-specific markers. Cells show ES-like morphology and highly expressed Oct4-EGFP (shows the establishment of endogenous ES cell signaling. In addition, OSKM / anti miR-29a iPS cells or OSK / anti miR-21 iPS cells And expressed ES cell-specific markers including SSEA1 and showed alkaline phosphatase activity To test whether these iPS cells show a pluripotent potential comparable to that of normally induced iPS cells, OSKM / anti miR-29a iPS cells and OSK / anti miR-21 iPS cells are induced to form embryoid bodies (EBs) or injected into nude mice to differentiate into various tissues. Two weeks later, typical cell types obtained from all three germ layers are observed. Histopathological analysis is performed on teratoma tumors formed after 3 weeks, confirming that various tissues from all 3 germ layers were generated and iPS cells gained pluripotency. To use the most stringent test for potency, iPS cells are injected into E3.5 blastocysts to create chimeric mice, mice obtained from iPS cells with miR depletion are due to iPS cells. Significantly about 15% to 25% black hair color These data indicate that depletion of miR-21 and miR-29a does not adversely affect the pluripotency of the resulting iPS cells .

Example 14
To understand the mechanism underlying the effect of miR-29a on reprogramming downregulation of P53 through the P85α and CDC42 pathways, the inhibition of miR-29a is investigated for expression of p85α and CDC42. p85α and CDC42 have been reported to be direct targets of miR-29 in HeLa cells. To that end, miRNA inhibitors are transfected into MEFs and p85α and CDC42 protein expression is assessed by Western blot 5 days after transfection. P85α and CDC42 protein levels are slightly increased by miR-29a blockade, whereas let-7a inhibitors have little effect (FIGS. 22a and 22b). In addition, transformation-related protein 53 (Trp53 or p53) has been reported to be a direct target of p85α and CDC4. Therefore, we investigate whether p53 is also regulated indirectly by miR-29a in MEF. To test it, MEFs are transfected with miRNA inhibitors and taken for 5 days for immunoblotting to assess p53 expression. P53 protein levels are reduced by approximately 30% by miR-29a inhibition (FIGS. 22a and 22b), but not altered by NT control or let-7a inhibition. It is important that depletion of miR-21 also releases p85α and CDC42 protein repression, thus increasing p85α and CDC42 levels, resulting in an approximately 25% down-regulation of p53 expression (FIGS. 22a and 22). 22b).

  To further confirm that inhibition of miR-21 or miR-29a reduces p53 levels during reprogramming, p53 expression is examined by Western blot analysis on day 5 of reprogramming. To initiate reprogramming, miRNA inhibitors are introduced with OSKM. Consistent with observations with MEF alone, depletion of miR-21 or miR-29a reduces p53 protein levels by about 25% or about 40%, respectively, compared to OSKM controls during reprogramming (FIG. 22c). In summary, our data showed that miR-29a blockade reduced p53 protein levels by approximately 30-40% through the p85α and CDC42 pathways during reprogramming. In addition, miR-21 depletion had a similar effect on both p85α and CDC42, reducing p53 protein levels by about 25% to about 30%.

  Inhibition of miR-29a improves initialization efficiency through p53 down-regulation: It has been reported that d53 deficiency can significantly improve initialization efficiency. According to the present invention, the effect of miR-29a knockdown is mainly mediated by p53 down-regulation, because miR-29a depletion significantly reduces p53 levels and improves initialization efficiency by about 2.8 times It will be. In response, p53 siRNA and / or miR-29a inhibitor is transfected into Oct4-EGFP MEF with OSKM to initiate reprogramming. Down-regulation of p53 by siRNA (approximately 80%) has a positive effect on reprogramming efficiency similar to miR-29a inhibition (FIG. 22d). When a miR inhibitor is added in the presence of p53 siRNA, no improvement in initialization efficiency is observed (FIG. 22d). These results suggest that miR-29a inhibition acts to some extent to improve reprogramming efficiency through down regulation of p53.

Example 15
Inhibition of miR-21 and miR-29a reduces ERK1 / 2 but not GSK3β phosphorylation and promotes reprogramming miR21 activates MAPK / ERK through inhibition of sproutey homologue 1 (Spry1) in cardiac fibroblasts It has been reported that Blocking MAPK / ERK activity promotes neural stem cell reprogramming and ensures a ground state for ESC self-renewal. Thus, according to the present invention, miR-21 regulates the MAPK / ERK pathway during initialization by evaluating ERK1 / 2 phosphorylation in MEF after miRNA inhibitor introduction. To test it, MEFs are transfected with miRNA inhibitors and then harvested for Western blot analysis to determine phosphorylated ERK1 / 2 levels. Western blot analysis showed that blockade of miR-21 significantly reduced ERK1 / 2 phosphorylation by approximately 45% compared to the NT control, whereas let-7a inhibitor had no such effect. (FIG. 23a). Interestingly, miR-29a depleted MEF also significantly reduces ERK1 / 2 phosphorylation by 60% compared to NT control (FIG. 23a). Also, according to the present invention, miR-21 and miR-29a can affect ERK1 / 2 phosphorylation by changing the Spry1 level. Transfecting with various miRNA inhibitors depleted miR-21 or miR-29a in MEF, and Spri1 expression levels were quantified by immunoblotting, and their results were expressed by inhibition of miR-21 and miR-29a. It shows that the expression level increased (FIG. 23b). Thus, depletion of miR-21 and miR-29a down-regulates ERK1 / 2 phosphorylation by modulating Spry1 protein levels.

  To address whether ERK1 / 2 downregulation increases initialization efficiency, siRNA targeting ERK1 or 2 is introduced into Oct4-EGFP MEF during the 4F initialization process. Any depletion promotes the generation of mature iPS cells (FIG. 23c). According to the present invention, miR-21 acts as an inducer of ERK1 / 2 activation in MEF because ERK1 / 2 phosphorylation is reduced by miR-21 blockade. Depletion of miR-29a also significantly reduces ERK1 / 2 phosphorylation. These results strongly suggest that miR-21 and miR-29a regulate ERK1 / 2 activity to increase reprogramming efficiency (FIGS. 23a, 23b and 23c).

  The GSK3β pathway also suppresses ES self-renewal and neural stem cell reprogramming. Depletion of GSK3β greatly increases mature iPS cell generation (FIG. 23c). According to the present invention, miRNA depletion modulated GSK3β activation. Immunoblotting shows that blocking miRNA in Oct4-EGFP MEF does not have a significant effect on GSK3β activation (FIG. 23d). According to the present invention, miRNA depletion will alter apoptosis or cell proliferation during reprogramming by using flow cytometry to assess cell viability and replication rate. Blockage of miRNA 21, 29a or let-7 during initialization with OSKM does not alter apoptosis or proliferation rate (FIG. 26). Overall, miR-29a and miR-21 modulate the p53 and ERK1 / 2 pathways to regulate iPS cell reprogramming efficiency.

Example 16
C-Myc is an alternative to the transgene currently used for reprogramming induction that lowers reprogramming threshold by lowering P53 levels and antagonizing ERK1 / 2 activation through downregulation of miR-21 and miR-29a It is important to understand how these factors regulate signal transduction pathways. According to the present invention, c-Myc will suppress miF-enriched miRNA (such as miR-21, let-7a, and miR-29a) during initialization (FIG. 20). Depletion of miR-29a by inhibitors is likely to reduce p53 protein levels by releasing p85α and CDC42 suppression (FIG. 22). In addition, miR-21 depletion also reduces ERK1 / 2 phosphorylation (FIG. 23). According to the present invention, miR-21 inhibition will reduce p53 protein levels, and miR-29a inhibition will also reduce ERK1 / 2 phosphorylation levels. Both p53 and ERK1 / 2 signaling antagonize reprogramming. Initialization efficiency can be increased by blocking miR-21 and miR-29a or knocking down p53 and ERK1 / 2 (FIGS. 22 and 23). According to the present invention, c-Myc is to some extent by inhibiting MEF-enriched miRNAs, miR-21 and miR-29a, which act as reprogramming barriers through induction of p53 protein levels and ERK1 / 2 activation. Initialization is promoted (FIG. 24).

  To facilitate reprogramming, forced expression of miR-290 family ES-specific miRNAs can be substituted for c-Myc. C-Myc binds to the promoter region of the miR-290 cluster. However, although early expression of the c-Myc transgene is effective to initiate reprogramming, it is not critical at the transitional stage of mature iPS cells where the miR-290 cluster begins expression or beyond. Thus, it is unlikely that c-Myc promotes the initial stages of initialization through activation of the miR-290 family.

  According to the present invention, when c-Myc is introduced for initialization, the expression level of MEF-enriched miRNA (including miR-29a, miR-21, miR-143 and let-7a) decreases. Will be. C-Myc has a severe transcriptional effect on miRNA in promoting tumorigenesis or maintaining pluripotent ground state. Therefore, c-Myc suppression of miRNA expression is a mechanism considered to be the basis of reprogramming.

  miR-21 acts as a positive mediator to enhance fibrogenic activity through the TGFβ1 and ERK1 / 2 pathways, both of which are known to affect reprogramming and ES cell ground state . In particular, among identified miR-29a targets, p53 is positively regulated by miR-29a. In addition, recent studies have shown that the Ink4-Arf / p53 / p21 pathway interferes with initialization and that p53 deficiency significantly increases initialization efficiency. From these, it can be said that these signaling pathways are the first barrier to the initialization process.

  Depletion of miRNA, miR-21 and miR-29a targeting c-Myc increases initialization efficiency by about 2.1 to about 2.8 times (FIG. 20) and miF-enriched miRNA is also an initialization barrier Is suggested to work as. It has recently been reported that let-7 inhibition promotes reprogramming, however, several attempts have shown that the effects on reprogramming observed are mild when antagomir inhibits let-7. It is only a thing (Figure 20). Furthermore, according to the present invention, the induction of p53 during initialization is hindered by miR-29a inhibition and the initialization efficiency is increased. Similarly, initialization can be greatly facilitated by either miR-21 depletion or ERK1 / 2. C-Myc is a major factor in the initial stage of initialization and is not necessary to maintain the process at the transitional stage and near the end of the process, in order to initiate a highly efficient initialization. miRNAs that regulate c-Myc could be used.

  C-Myc has been reported to bind and repress directly with the miR-29a promoter. According to the present invention, c-Myc can only be partially replaced by miR-21 depletion, suggesting that c-Myc has other functions in initialization. Thus, instead of c-Myc function during initialization, multiple pathway regulation or broad suppression of MEF enriched miRNAs may be required.

  According to the present invention, c-Myc will lower the threshold of initialization by lowering p53 levels and antagonizing ERK1 / 2 activation through miR-21 and miR-29a down-regulation. In addition, factors downstream of c-Myc can be subject to manipulation with siRNA, miRNA or small molecules to improve reprogramming. These approaches can be extended to replace all four reprogramming factors.

  While the invention has been described by way of the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims (39)

A method of generating induced pluripotent stem (iPS) cells, comprising:
contacting a somatic cell with a nuclear reprogramming factor; and b) contacting the cell of (a) with a microRNA that alters RNA levels or activity within the cell, thereby generating an iPS cell. The method of generating comprising.   The method of claim 1, wherein the microRNA or RNA is modified.   The method of claim 1, wherein the microRNA is present in a vector.   2. The method of claim 1, wherein the microRNA is present in a miR-17, miR-25, miR-106a, miR let-7 family member or miR-302b cluster.   2. The method of claim 1, wherein the microRNA is miR-93, miR-106b, miR-21, miR-29a, or a combination thereof.   The method of claim 1, wherein the microRNA has a polynucleotide sequence comprising SEQ ID NO: 1.   The method of claim 1, wherein the microRNA has a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 2-11.   2. The method of claim 1, wherein the microRNA modulates the expression or activity of p21, Tgfbr2, p53, Ago2, or a combination thereof.   2. The method of claim 1, wherein the microRNA modulates the Splay 1/2, p85, CDC42 or ERK1 / 2 pathway.   The method of claim 1, wherein the nuclear reprogramming factor is encoded by a gene contained in a vector.   The method according to claim 1, wherein the nuclear reprogramming factor is a SOX family gene, a KLF family gene, a MYC family gene, SALL4, OCT4, NANOG, LIN28, or a combination thereof.   The method according to claim 1, wherein the nuclear reprogramming factor is one or more of OCT4, SOX2, KLF4, and C-MYC.   The method of claim 1, wherein the nuclear reprogramming factor comprises c-Myc.   2. The method of claim 1, wherein the reprogramming factor is contacted with the somatic cell before, simultaneously with, or after contact with the microRNA.   2. The method of claim 1, wherein the somatic cell is a mammalian cell.   Induced pluripotent stem (iPS) cells produced using the method of claim 1.   An enriched population of induced pluripotent stem (iPS) cells produced by the method of claim 1.   A differentiated cell obtained by inducing differentiation of a pluripotent stem cell produced by the method according to claim 1. A method of treating a subject comprising:
a) generating induced pluripotent stem (iPS) cells from the subject's somatic cells by the method of claim 1;
b) inducing differentiation of the iPS cells of step (a); and c) introducing the cells of (b) into the subject, thereby treating the condition, said method of treatment.   Use of microRNA to enhance iPS cell production efficiency.   The microRNA is from miR-17, miR-25, miR-93, miR-106a, miR-106b, miR-21, miR-29a, miR-302b cluster, miR let-7 family member, or combinations thereof 21. Use according to claim 20, wherein the use is selected from the group consisting of:   from at least two or more of miR-17, miR-25, miR-93, miR-106a, miR-106b, miR-21, miR-29a, miR-302b cluster, miR let-7 family member, or combinations thereof A combination of miR sequences selected from the group consisting of: A method of generating induced pluripotent stem (iPS) cells, comprising:
a) contacting a somatic cell with a nuclear reprogramming factor; and b) contacting the cell of (a) with an inhibitor of a microRNA, thereby generating iPS cells.   24. The method of claim 23, wherein the microRNA is present in a miR-17, miR-25, miR-106a, miR let-7 family member, or miR-302b cluster.   24. The method of claim 23, wherein the microRNA is miR-93, miR-106b, miR-21, miR-29a, or a combination thereof.   24. The method of claim 23, wherein the microRNA has a polynucleotide sequence set forth in SEQ ID NO: 1.   24. The method of claim 23, wherein the microRNA has a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 2-11.   24. The method of claim 23, wherein the microRNA modulates the expression or activity of p21, Tgfbr2, p53, Ago2, or a combination thereof.   24. The method of claim 23, wherein the microRNA modulates a Splay 1/2, p85, CDC42, or ERK1 / 2 pathway.   24. The method of claim 23, wherein the nuclear reprogramming factor is encoded by a gene contained in a vector.   24. The method of claim 23, wherein the nuclear reprogramming factor is a SOX family gene, a KLF family gene, a MYC family gene, SALL4, OCT4, NANOG, LIN28, or a combination thereof.   The method according to claim 23, wherein the nuclear reprogramming factor is one or more of OCT4, SOX2, KLF4, and C-MYC.   24. The method of claim 23, wherein the initialization efficiency is at least twice compared to a control that does not use the microRNA inhibitor.   24. The method of claim 23, wherein the somatic cells comprise fibroblasts.   24. The method of claim 23, wherein the somatic cell is contacted with the reprogramming factor before, simultaneously with, or after contact with the microRNA inhibitor.   24. The method of claim 23, wherein the somatic cell is a mammalian cell.   24. Induced pluripotent stem (iPS) cells produced using the method of claim 23.   24. An enriched population of induced pluripotent stem (iPS) cells produced by the method of claim 23.   A differentiated cell obtained by inducing differentiation of the pluripotent stem cell according to claim 38.

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